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					Journal of Histochemistry & Cytochemistry
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 DYEBINDING AND MUCOPOLYSACCHARIDE CONTENT IN CONNECTIVE TISSUE
                           ENDRE A. BALAZS and JOHN A. SZIRMAI
                             J Histochem Cytochem 1958 6: 416
                                    DOI: 10.1177/6.6.416

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                            http://jhc.sagepub.com/content/6/6/416


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                       DYEBINDING                             AND                MUCOPOLYSACCHARIDE                                                      CONTENT
                                                                    IN         CONNECTIVE                           TISSUE’

                                                 ENDRE                   A.     BALAZS           AND     JOHN                A.      SZIRMAI2
   From       the Retina          Foundation,                 Department         of Ophthalmology         of the Massachusetts                                                    Eye         and    Ear       Infirmary
                                           and             Harvard       Medical     School,      Boston,    Massachusetts


   The      uptake         of cationic              dyes      by         proteins,    nucleic which                    the           relationship                       between               the        mucopoly-
acids   and          other    macromolecules                         has        been  studied saccharide                            content                 of     the          mucoid              layer          of       the
to estimate              the    number       of             anionic             sites (13,     rooster
                                                                                               15,                      comb              and         that         of     the      hyaline             cartilage              is
23).   More             complex        systems,                  such            as cellulose, compared                           with          the          dyebinding.                  The         dyebinding
cotton         and        wool          fibers        (22),fibrin             films    (19),           of the        fresh           tissue,           the        water-extracted                        tissue         and
bacteria       (10),         yeast          cells      (23)         and          viruses             the
                                                                                                  (17),        water              extract             was         compared                with        the         respec-
were     also used         for    the       quantitative            determination                    tive       mucopolysaccharide                               contents              of these           tissues.
of cationic             dye   uptake.                 Several       attempts                     have
                                                                                                                                   MATERIAL                      AND METHODS
been    made            to correlate                 the     amount         of              cationic
dye bound      by a tissue       with   its nucleic      acid                               content     1. Preparation       of tissues: Mucoid         layer    was
(20).    In connective       tissues      it was        that
                                                    found                                  the      obtained     from  the comb     of White      Leghorn      roosters
staining    of tissue  sections       with   basic    dyes                                 depends (6 to 18 months         old)  and    dissected         as described
                                                                                                       previously                  (6).     Cross            sections             of    the      whole         comb           of
upon    the pH of                the       dye       solution        used             (8).
                                                                                       7-week-old           White        Leghorn           cockerels           treated        with
   Mucopolysaccharides                           and         various                  sulfated
                                                                                        varying        amounts            of testosterone                 also      were      used.
polysaccharides                can       be precipitated              from        aque-
                                                                                        Hyaline        cartilage          was taken            from     the septum             nasi
ous     solutions         by       cationic       dyes.        The      precipitate
                                                                                        of steers         (2 to 3 years                old)      and     prepared           as de-
formed        represents          a complex         between          the dye and        scribed        (6).     The       fresh       tissues        were        sectioned          in
the polysaccharide                  in which       the amount              of bound various           experiments               to a thickness               of 15 to 100
dye and the number                    of anionic       sites are in stoichio- on a freezing                      microtome.             Samples        of measured              wet
metric       relation         (5).      A method            for the        quantita- weight           were       used        to determine               the       dyebinding,
tive    determination                of the amount              of cationic             the
                                                                                      dye       hexosamine             content,           and      the    water         content,
bound         by various              connective          tissues        has      previ-as well         as for        the       preparation            of mucopolysac-
ously       been       described            by   the      authors           (6).        charides
                                                                                     This               and for extraction                    with     water.
                                                                                             For water         extraction           20 to 1000 mg (wet                   weight)
method         was used          to determine            the      dyebinding              in
                                                                                                       of    frozen           sectioned                  tissue           was          placed            in   varying
various     connective                 tissues         (3, 4, 21).               was found
                                                                                 It
                                                                                               volumes          (50 to 500 ml) of distilled                        water,        with
that    dyebinding                   can       be       completely              suppressed
                                                                                               continuous            mechanical             agitation            at +4#{176}C      for 1 to
by      increasing               the       hydrogen              ion     concentration.        7 days.          The     tissue       fragments                 were
                                                                                                                                                                 separated              by
This       suggests          that       the       interaction            between           the centrifugation              for 1 hr 2500     at           x G or,         in certain
tissue        and      the        dye     is electrostatic                in nature           and
                                                                                               cases,       for 1 to 2 hrs at          100,000           x G. If tissue            f rag-
the      anionic           sites      of the           tissue       are     primarily          ments
                                                                                                re-          accumulated            on the surface                    of the solution,
sponsible            for the          dyebinding.               The      fact      that        they
                                                                                             con-         were      removed           by filtration                 through            several
nective         tissues         containing              large     amounts           of muco- layers          of cheesecloth               in the            case      of comb             tissue
polysaccharides                    show      a high           dyebinding            capacity and        through        Whatman                    1
                                                                                                                                              filter       paper        in the case
                                                                                               of cartilage.           This      clear       supernatant                   was      dialyzed
suggests           a possible            correlation            between          the muco-
                                                                                               in Nojax           Visking        casings             against           distilled          water
polysaccharide                   content         of tissue and its cationic
                                                        a
                                                                                               for 24 hrs            at +4#{176}C     under           constant             shaking           and
dyebinding.                                                                                    used       for     determinations                   of dyebinding,                     hexosa-
     This      paper          describes           a series         of experiments               in
                                                                                               mine,         hexuronic          acid        and         total        nitrogen            or for
     ‘This         investigation               was supported                 in part           mucopolysaccharide
                                                                                            by a                                  preparation.                   In some              cases
research           grant         (A-1110)         from      the      National          dyebinding
                                                                                       In-                   also was     determined           on      undialyzed
stitute        of Arthritis           and Metabolic               Diseases       of theextracts.
National             Institutes           of      Health,          Public       Health
                                                                                           The     residue       of the       water-extracted       tissue     was
Service,          and      by the        Trustees         of the        Godfrey         M.
Hyams           Trust.        Paper       63, Retina           Foundation.              used for dyebinding              experiments          after      the compact
     2 Present          address:        Research           Laboratories,             De-
partment            of Rheumatology,                   University          Hospital,         Perandren           Propionate,            Ciba      Pharmaceutical
Leiden,          Netherlands.                                                          Products,          Inc.,    Summit,         N. J.
                                                                                                 416




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                    DYEBINDING                     AND            MUCOPOLYSACCHARIDE                                   CONTENT                 IN       CONNECTIVE                    TISSUE                      417


tissue         pellet         collected              from          the       bottom           of the            was dissolved
                                                                                                             cen-                              in the solution.                        Thymol              crystals               or
 trifuge          tube        had        been         resectioned                  on the freezing 1 ml 1% merthiolate                                             was added                  to 100 ml of the
 microtome.                 The         same           material               was       used         for       solution
                                                                                                              the                 to prevent                bacterial              growth,            and the solu-
 preparation                 of mucopolysaccharides                              and       for       the        tion      was then              dialyzed                against           4 to 5 liters                     M/15
 determination                     of its water                   content.                                      phosphate             buffer          at pH 7.2 for 2 days                           at 37#{176}C,         with
      2. Determination                          of       the           dyebinding            : The              several         changes            of the outside                     fluid.         At the end of
 dyebinding                 of the            fresh         and          the      water-extracted               this      procedure                both           the       cartilage              and          the         comb
 tissues          was       determined                     as described (6) using                       25 tissue             sections            were           completely                 digested,                leaving
 to 100 ml of 1 to 4 X 10 M azure                                                   A solutions                 a somewhat                 cloudy             solution.             Trichloroacetic                          acid
  (pH      6 to 7).4 The                tissue          sections              were       15 to ,100 was                  then        added           to the solution                       to a final                 concen-
 thick.                                                                                                         tration         of 10%. After                    thorough              mixing           the solution
      To determine                     the        dyebinding                   in the          water          ex-
                                                                                                                was stored             overnight                 at +4#{176}C,       and the precipitate
 tract,        various            volumes               of extract                (1 to 5 ml) were             formed           was separated                       by filtration               through                 What-
 added          to 5 to 20 ml of 1 to 4 X M azure                                   10                  A       man            1 filter        paper.            The         mucopolysaccharides                      of
 solutions              (pH        6 to 7). The                       solutions             were         main-the clear             supernatant                    were then               precipitated                     with
 tained                           for
                at +4#{176}C 24hrs under                              constant           mechani-              4 volumes               of absolute                    ethanol            while         the         solution
 cal agitation                and werethencentrifugedat                                2500      X g            was maintained                      at +4#{176}C          for 16 hrs,              during              which
 for 20 mm at room                         temperature                    in order         to remove time                  the precipitate                       settled           at the bottom                          of the
 the precipitate.                    The          supernatant                   was diluted                withcontainer.             The supernatant                          was decanted,                         and the
 distilled          water          (6 to 21 X),                  and the optical                    density sediment               was      collected                by centrifugation                            at
                                                                                                                                                                                                                 2500
 was       measured                at 620 m.i using                     cells      with         a light X g for 15 mm                              and          washed           3 times             with          absolute
 path         of 1 cm in a Beckman                                          B or DU                 spectro- ethanol.              The sediment                       was then              dried        to constant
 photometer.                    In        some           cases            absorption                 spectra weight              at     80#{176}C     in vacuum.                      Determinations                   of
 were        recorded             between               500 and 650 m.                                          hexosamine,                hexuronic                  acid      and ester              sulfate              were
      3. Estimation                        of mucopolysaccharide                               con-             made         either        on the dry                     powder           or on known                         vol-
 tent:         The         mucopolysaccharide                             content             of the umes                    of the          solution                prior        to precipitation                           with
 fresh        tissue,          the extracted                        tissue,          and       the       tissueethanol.            The       total         mucopolysaccharide                     content               of
 extract           was        calculated                   from             the        hexosamine,              the tissue,           the tissue               extract          or the extracted                         tissue
 hexuronic               acid,        and         ester         sulfate           determinations.               residue           was       calculated                   from         the       hexuronic                    acid
 These          were        carried             out       on the tissue                   sections,              on
                                                                                                                and      the ester             sulfate             determinations.
 the       extract           itself,           or on the                   pure        mucopolysac-                  Hexosamine,                 hexuronic                  acid,        nitrogen                and         sul-
 charide           prepared               from         them.                                                    fur analyses                were         made            in order            to determine                       the
       Preparation               of mucopolysaccharides.                           Hyaluronic                   purity         of the prepared                         mucopolysaccharides.                      The
 acid       and        chondroitin                  sulfuric              acid       were        prepared glucosamine                      and        galactosamine              contents              were also
 from         comb          and         cartilage                by the            proteolytic                 en-
                                                                                                                determined.              Typical               analysis            values         obtained                 from
 zyme         digestion             method.               Pepsin            and trypsin                 diges- mucopolysaccharide                         fractions              prepared              as sodium
 tion      was suggested                      by Hadidian                     and Pirie             (12) for    salt      are as follows: Cartilage:                           hexosamine                 33.8%,
 the purification                    of hyaluronic                     acid of the umbilical hexuronic                                acid 37.1%,                   sulfur         5.0%,        nitrogen                  2.1%,
 cord.        A modification                       of this            method was used               for         galactosamine                   99% and glucosamine                                  1.0%           of total
 the quantitative                        recovery              of hyaluronic                   acid       fromhexosamine                recovered.                Comb7:        hexosamine                    42.0%,
 the      rooster          comb            and the vitreous                          body        (2). This      hexuronic              acid 48.3%,                 sulfur           < 0.01%,                  nitrogen
 technique               does        not         allow          separation               of the            vari-
                                                                                                               3.15%,            galactosamine                         < 0.1%               and         glucosamine
 ous mucopolysaccharides                               but. yields              a protein-free                  100%       of total          hexosamine                    recovered.
 preparation                with          recovery              of large           molecular               non- Hyaluronidase                         digestion.          Testicular                 hyaluroni-
 dialyzable              mucopolysaccharides                         from these             tissues.            das&,         1.2 mg per 100 l extract, was used
                                                                                                                                                            m                                               for di-
      One       gram          (wet          weight)            of sectioned                 tissue
                                                                                                 was            gestion          in pH 5.3 acetate                            buffer          with         continuous
 placed         in 20 ml 0.1N HC1 containing                                      2 mg pepsin.5                dialysis            against             the        same          buffer          at 37#{176}C           for 27
 This        suspension            was placed                  in a dialysis                 bag and hrs. 1% merthiolate                                       (see above) was used                       as bac-
 dialyzed             against           4 to 5 liters                  N
                                                                       0.1 HC1 for 2 to 3 teriostatic                                  agent.            The         digested            extracts               were          dia-
 days         at 37#{176}C   with           several             changes              of the           fluid. lyzed           against            distilled              water           for 48 hrs prior                            to
 The       contents             of the dialysis                       bag were             neutralized         dyebinding               experiments.
 with        1 N sodium                hydroxide,                  and 2 mg of trypsin6
                                                                                                                       The preparation                     was          made          from          the mucoid
         National              Aniline               Division,                Lot                NAz          15,
                                                                                                               layer          of the            comb              of White                  Leghorn                   roosters
dye content                  92%.                                                                              approximately                    one year               old. The mucoid                         layer          was
         Pepsin,          2 X crystallized                         from         ethanol,           Worth- carefully                 separated               from the          central           core         and          the
ington          Biochemical                   Corp.,          Freehold,               N. J.                    epithelium.
       Trypsin,
      #{149}                crystallized,                    lyophilized,                  Worthing-                 8 Wydase,           1,500          USP             (TR)          units           per          10 ml,
ton       Biochemical                   Corp.,           Freehold,                N. J.                        Wyeth            Laboratories,                       Inc.,        Philadelphia,                        Pa.




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 418                                                     ENDRE              A.      BALAZS              AND JOHN              A. SZIRMAI


                                                                                                         added         to different            volumes          of 1 to 4 X M              10
                                                                                                         azure         A solutions.               Depending             on      the        volume
                                                                                                         added,            either        a    reddish-blue              precipitate              was
                                                                                                         formed           or an immediate                   metachromatic                  change
                                                                                                         took      place       in the solution.            The addition              of a small
                                                                                                         volume           of extract           led    to precipitate               formation,
                                                                                                         and      the absorption                curve      of the supernatant                   was
                                                                                                         similar       to that          of a pure           dye     solution           (Fig.      1).
                                                                                                         When         larger        volumes         of extract          were        added,         no
L
                                                                                                         precipitate              was formed          and     the absorption                 maxi-
‘I)
                                                                                                         mum         shifted         to     shorter         wavelengths                (Fig.        1,
                                                                                                         Curve        E) . Hexuronic           acid determinations                   on these
-4                                                                                                       extracts            indicate              that        a     metachromatic           shift        of
                                                                                                         the dye          solution             occurs          when         the       hexuronic                acid
                                                                                                         content          of the             added          extract             reaches             a certain
                                                                                                       amount.          The curves                obtained            in these            experiments
                                                                                                       are      very          similar            to those              noted            previously                for
                                                                                                      mucopolysaccharides                          and for tissues (5, 6).
                                                                                                           Based          on these observations                       the amount                  of dye
                                                                                                       bound          by a certain                   volume            of tissue             extract            was
                                                                                                      determined                   by measuring                     the       optical           density             of
                                                                                                       the     free         dye        remaining               in the           solution             after         re-
                                                                                                       moval          of the            precipitate.                The         amount              of bound
              500                    550                       600                       650
                                   WAVELENGTH-                 mj
                                                                                                       dye      in the precipitate                         was        calculated               from          these
                                                                                                       optical         density             measurements,                    as described                  in the
     F:o.      1. Effect           of cartilage                  extracts             on the          ab-
 sorption         spectrum             of the dyebath.                      5 ml of various previous                      paper         (6).      It was           found          that        there          is a
 cartilage         extracts          (seeTable            VII,       Exp.        15) were              linear         relationship                   between              the        hexuronic                 acid
 added        to 10 ml of azure                      A solution;                  the final          dye
                                                                                                       content             of the            extract            and        the       amount               of dye
 concentration             was      2.66 X 10                    M. The              solutions
                                                                                                       bound          in the           precipitate                (Fig.       2). Within                 a con-
 were      diluted          6 X with                  distilled            water           prior        to
 recording          the absorption                   curve.                                            siderable             range          of dye concentration                             and       extract
                                                                                                       volume            the        hexuronic              acid-bound                   dye       ratio         was
                                                                                                      constant.               The        cartilage            extract           bound           more          than
     Separation              of hexosamines.                 Glucosamine                      and
                                                                                                      twice        as much                dye      per micromole                hexuronic               acid
 galactosamine                were       separated                on an ion-exchange
                                                                                                      as did the comb                         extract.
 column           (Dowex            50, H cycle)                       according               to    the
                                                                                                           2. Dyebinding                         and            mucopolysaccharide
 method          described           by Gardell                 (11).
                                                                                                      content               in the            comb: The            mucoid            layer        ofthe
     Hexosamine             was        determined                     by        a modified
                                                                                                      comb         of roosters                 (6 to 18 months                     old)      was used               to
 Elson-Morgan                  method            (7).       When            tissue          sections
                                                                                                      determine                the amount                 of dye bound                     by the unex-
 were      used,      they       were       first extracted                    in acetone            for
                                                                                                      tracted           tissue,          the water-extracted                           tissue         and        the
 several        days       to remove                the       lipids         and        small       mo-
                                                                                                      water          extract.              The        results          of these              experiments
 lecular         components                 (glucose,              amino            acids,        etc.)
                                                                                                      are summarized                       in Table             I. The dyebinding                          of the
 which         might          interfere              with           the         determination
                                                                                                      isolated             mucoid              layer         shows           little         variation                in
  (14).
                                                                                                      animals            of this          age group.               It can also be seen that
     Hexuronic           acid was        determined                  according               to the
                                                                                                      when         this        tissue         is extracted                with        distilled             water
 carbazole          method           of Dische               (9). This             method          was
                                                                                                      the bulk              of the material                      responsible                for the dye-
 used       only       on dialyzed                tissue           extracts             or on the
                                                                                                      binding           is present               in the extract. The                    amount             of
 purified        mucopolysaccharide                    fraction.
                                                                                                      dye bound                 by the water             extract shows                  some         vari-
      The ester sulfate            content           of mucopolysaccharide
                                                                                                      ation,        depending                   on the           conditions                of water               ex-
 preparations                was        determined                    according                to the
                                                                                                      traction.             The       amount            of dye bound                    by the residue
 benzidine           sulfate         method              as modified                   by Ander-
                                                                                                      of the          water-extracted                       mucoid             layer         is less          than
 sen (1).
                                                                                                       10% of that bound                         by the originaltissue.
     The nitrogen            content           of dialyzed                 extracts            or of
                                                                                                           Similar          experiments                were         carried          out using       the
 mucopolysaccharide                   preparations                   was determined
                                                                                                       whole         comb           of cockerels                  (7 weeks              old)       following
 by the micro-Kjeldahl                         method              (16).
                                                                                                       18 days            of testosterone                     treatment               (Table            II).       As
                                                                                                      expected,              treatment           with      testosterone                   results         in a
                                      RESULTS
                                                                                                       considerable                 increase in the comb                       weight.           For the
     1. Dyebinding                of      extracts:             Various             volumes            largest         daily          dose        of testosterone                      the      amount               of
 of water         extracts         of comb            and cartilage                  tissue       were dye       bound,              calculated               for       the       total         amount                of




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                   DYEBINDING                  AND      MUCOPOLYSACCHARIDE                               CONTENT                   IN        CONNECTIVE                TISSUE                             419


comb       tissue,         shows       an increase           of more         than      ten
                                                                                        can       be observed                   in the        dyebinding                  of the          water
times       that       of the         control.        This      increase         is onlyextracts.           The        amount            of dye bound                   by the water-
twice      as great when             the     dyebinding            is expressed         extracted               tissue,             while        slightly              larger          in        the
in terms         of wet tissue              weight,        indicating          that     al-
                                                                                        treated         comb         than         in the control,                 varies       very        little
though          the      total        amount          of the         substance            re-
                                                                                        with       the level            of treatment.                 In the comb                 of young
sponsible           for the         dyebinding            increases         consider- treated           animals,             just      as in adult              roosters,         the        bulk
ably      during          treatment,            its concentration                 in the the material
                                                                                        of                                  responsible               for the dyebinding                           is
tissue      shows        a smaller          increase.        A similar          picture water-extractable.                     In both             the        treated           and         the
                                                                                        untreated              comb           of young             animals the           water-ex-
                                                                                        tracted            cross         section           binds          more          dye       than           the
                                                                                        water-extracted                   mucoid            layer          of the          full-grown
                                                                                        animal.            This         probably             can        be explained                  by the
                                                                                        difference            in the cell content                        of the two extracted
                                                                                        tissue        samples:             The        epithelium               is included              in the
                                                                                        cross        section           of the          comb          of young              animals              but
                                                                                        not       in the         sections            of the          mucoid            layer        of adult
                                                                                        animals.            Also,        the connective                   tissue        of the central
                                                                                        core and of the mucoid                          layer       of young            animals           con-
                                                                                         tains      more         cells      than        that      of the adult                animals.
                                                                                              Among           other          factors,          the       effectiveness                of the
                                                                                         water       extraction               is influenced              by the ratio              of tissue
C-)
                                                                                         to extracting                  fluid.       When          various            weights           of the
                                                                                        mucoid            layer         sections           are extracted                   in the           same
                                                                                         volume            of distilled                  water           (200        ml),        the         total

                                                                                                                                             TABLE              I
                                                                                                   Dyebinding               in     the Mucoid               Layer            of the Rooster                          Comb
                                                                                                      In  Exps.       14 and       16 the     amount    of sectioned
                                                                                                   mucoid      layer     extracted         with 100 ml  distilled
                                                                                                   water  was 25 to 520 mg;          in Exp. 36 one gram;        and
                                                                                                   2 g in Exp.       30.

                                                                                                                                Dye  Bound               Dye     Bound              Dye     Bound           in
                                                                                                                Exp.         * per Gram                     in Water               Water-Extracted
                                                                                                                                           of
                                                                                                                                    Tissue               Extract     of        1    Residue       of        g
                                                                                                                                                                                                            1
                          0.2                  0.4               0.6                0.8                                                                     g Tissue                      Tissue

                            HEXURON1C                   ACID-       MICROMOLES                       months                         pmoles                  smo1es                        Mmoles
   FIG.       2.     Relationship                    betweenamount       of dye
                                                                                                 6                     16                    36.3            35.1                                -
bound         and       hexuronic              acid    content     in extracts                of
cartilage              and     mucoid             layer of the     comb.     For
                                                                                                 6                     30                    33.8            24.5                             2.1
further            data     see Tables                 III   and           VII.    Hexuronic     9                     36                    33.1            32.9                             1.8
acid was            determined        on             samples             of dialyzed        ex- 18                     14                    35.2            27.5                                -

tracts.

                                                                                      TABLE              II
                                               Dyebinding                in   the    comb     of     testosterone-treated                       cockerels
     Dyebinding              was          determined               on      whole       cross     sections              of the   comb                  of 7-week-old        cockerels                                     injected
intramuscularly               with          testosterone                 propionate          daily      for        18 days.      The                remainder       of the     comb                                    was sec-
tioned       and       extracted              in distilled              water       (20 ml per gram                 of tissue).
                                                                                Micromoles         Dye Bound  in the Comb               of    Micromoles             Dye Bound          per      Gram           of
                             .                                                                     One Animal                                                          Comb
                         Daily   Tstosterone
       Exp.                                              Comb      Weight
                                                                                    Whole            Water             Extracted                Whole                Water                    Extracted
                                                                                    tissue           extract             residue                tissue               extract                    residue

                                     mg                            g

       27-A                      1000                           16.49               328.2            272.1                  52.8                 19.9                 16.5                           3.2
       27-B                       250                           11.08               175.1            151.8                  39.9                 15.8                 13.7                           3.6
       27-D                        25                            5.78                93.6             69.4                  20.8                 16.2                 12.0                           3.6
       27-E                          -                           2.75                28.1             11.8                   6.9                 10.2                  4.3                           2.5




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420                                                        ENDRE            A.       BALAZS              AND            JOHN             A.       SZIRMAI



amount             of hyaluronic                     acid        recovered                in the          ex- The            mucopolysaccharide                      fraction                was            pre-
tract       increases            with        the tissue              weight,            but extrac- pared                 from        the        water-extracted               residue           and          its
tion       is most              effective             when          the         smallest             tissuehexosamine             and hexuronic                    acid        content           compared
weight         is used         (Table          III).       However,                the hexuronic with                  the       dyebinding                 values            (Table           IV).          In the
acid      content           of the extract                   and the amount                        of dye  tissue        residue          of the comb               sections            the molar                ratio
bound           are      proportional                   in all extracts.                      Further- of the bound                      dye to the hexuronic                            acid       is slightly
more,         it appears               that         the       ratio       of micromoles                    more
                                                                                                             of         than        one,       indicating              that         20 to 30%                  of the
bound          dye to micromoles                          of hexuronic                   acid in the       bound         dye       in the          extracted              tissue          cannot            be at-
extract          shows          an average                value        of 1.07 in the four                 tributed             to the           hyaluronic                acid        present.              In the
extracts,            suggesting               a stoichiometric                        relationship         residue           of the          mucoid           layer,          however,              the        molar
between            the number                 of anionic              sites         of hyaluronic ratio                  of the            bound           dye        to        the      hexuronic                   acid
acid      in the extract                     and       the       number              of dye mole-          present           is slightly            less than               one.       (Exp.         30, Table
cules       bound.                                                                                         IV.)
                                                                                                                The hexosamine                    and       hexuronic               acid contents                   of
                                                                                                           the water              extract          ofthe       comb          sections             (Exp.           27
                                     TABLE                III
                                                                                                           A-D)        were        determined,                  and         pure        mucopolysac-
Hyaluronic                Acid          Content            and        Dyebinding                   in the
                                                                                                           charide           was       prepared             from         the        mucoid            layer           ex-
               Water         Extract           of the Mucoid                      Layer
                                                                                                           tracts         (Exp.         16, 30).         (Table            IV.)       It is interesting
     Various            amounts                 of sectioned                       mucoid             layerto note          that       in Exp.          27 the hexosamine-hexuronic
were         extracted               with         200 ml             of distilled                  water. acid       molar         ratio       is more          than         one, suggesting                      that
Dyebinding,                 hexosamine                   and hexuronic                     acid       weresome           glycoproteins                   which           contain              hexosamines
determined                 on dialyzed                   samples               of the           extract. are present                 in this          crude         extract.             In the            purified
Hyaluronic                 acid        was         estimated                on the              basis mucopolysaccharide
                                                                                                              of                                   fraction            of Exp.           30 the hexo-
hexuronic              acid       content.                                                                 samine-hexuronic                       acid        ratio         is one. he T           bound
                                                                                                           dye-hexuronic                    acid      molar          ratio          gives        an average
                                                               Dye          Hyalu-
                                                                                                           of 1.01           in all experiments                          without             showing                 any
                            Hexos-       Hexuron- Bound      Water in       ronic in
                                                                            Acid
                Ex-        amine in         ic Acid         Extract           Water           (1)          significant             difference             between              comb         extracts                and
Exp.       * tracted                                           of 1        Extract
                             200 ml      in 200 ml                                                         mucoid           layer        extracts.
              Tissue        Extract        Extract            gram        of 1 gram
                                                             Tissue          Tissue                             The       hexuronic             acid      and hexosamine                        contents               of
                                                                (1)              (2)
                                                                                                           the      purified           mucopolysaccharide                      fraction             of the
                 mg         imoies          pmotes          mtmoles          .imoles                       unextracted                  mucoid             layer          sections             were            deter-
                                                                                                           mined           (Table          V). The             bound           dye-hexuronic                       acid
  16-A           107.4 3.58                   4.76             41.9            44.8         0.94
                                                                                                          molar          ratio       is more           than        one,        indicating                that         the
  16-B           272.8 8.26                   7.92             33.3            28.5          1.16
                                                                                                           amount             of dye         bound,           as thein         extracted               tissue
  16-C         509.9        16.52           14.20              32.1            27.4          1.16
                                                                                                           residue,           is 15 to 25% more                     than         would         be expected
  16-D        1043.6 35.64                  27.72              26.6            26.0          1.02
                                                                                                          from       the hyaluronic                    acid       content.

                                                                                              TABLE                IV
        Mucopolysaccharide                         Content            and    Dyebinding                of the           Water           Extract         and        the    Extracted             Residue            of
                                                                                               the    Comb
    In Exps. 16 and           30 isolated        mucoid         layer       of rooster       comb      was    used      (Table          I).    In Exp.        27   the      whole
comb       of testosterone-treated             cockerels          was used          (Table      II).   In the water           extracts           of Exps.        16 and       27
the hexosamine               and     hexuronic          acid       contents        were     determined         on dialyzed             samples.         In the        water-ex-
tracted       residue       of the tissue         and in the extract                 of Exp.      30, these    determinations                 were made          on the poly-
saccharide          prepared       by enzyme            digestion.          In Exp.       16 the values      are an average                 of four    different       extracts
 (Table     III).

                                                Water      Extract         1
                                                                        of grain    of Tissue                                    Water-Extracted                Residue     of        1 gram   of Tissue

      Exp.      *                                                                                            (1)               Bound                                      Hexuronic                      (3)
                            Bound         dye                               Hezuronic          acid
                                                        Hexosainine                                                             dye                Hexosamine                    acid
                                    (1)                                                 (2)                  (2)                  (3)                                             (4)                )

                               mimoles                     pmoles                  wnoles                                      pmoles                imoles                  .smoles

        16                      33.4                        32.3                    31.7                  1.05                    -                     -                         -                  -


        30                      24.5                        21.1                    21.8                  1.12                   2.1                   4.2                       2.5               0.84
        27-A                    16.5                        20.3                    17.0                  0.98                   3.2                   3.0                       2.4               1.33
        27-B                    13.7                        21.2                    13.5                  1.01                   3.6                   4.3                       3.2               1.13
        27-D                    12.0                        16.4                    12.1                  0.99                   3.6                   7.4                       2.8               1.29




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                 DYEBINDING                  AND        MUCOPOLYSACCHARIDE                             CONTENT                  IN         CONNECTIVE                       TISSUE                   421


                                      TABLE             V                                                                                TABLE                     VI
Mucopolysaccharide                    Content         and Dyebinding      of the                      Mucopolysaccharide                              Content               and         Dyebinding
        Mucoid               Layer         of       the   Rooster    Comb                                                                in           Cartilage
                                                                                               1.8 to 1.9 g of cartilage                  tissue was extracted               with
    In     Exp.          16 the         hexosamine              content           was     de-
                                                                                           100 ml of distilled                water.        In the tissue,               the hexos-
termined           directly          on the tissue;             in all other           cases
the      hexosamine               and      the   hexuronic            acid       contents amine,          hexuronic            acid      and       sulfur        contents           were
                                                                                           determined              on the           polysaccharide                 prepared             by
were       determined               on the      polysaccharide                 prepared
                                                                                           enzyme         digestion.           In Exp.          31 the hexosamine                    and
by enzyme               digestion.
                                                                                           hexuronic           acid       contents          were        determined              on the
                 Dye      Bound
                                                      Hexuronic                            dialyzed          extract.          All sulfur             determinations                  and
                                     Hexosamine        Acid      per         (1)
   Exp.    *    cram            of per Gram      of    Gram       of                       hexosamine             and      hexuronic            acid      determinations                of
                                        Tissue          Tissue               (2)
                        (1)
                                                            (2)
                                                                                           the      other        extracts           were        made         on     the       polysac-
                                                                                           charide          prepared            from         the      extract           by      enzyme
                    .imoles             emoles          moles
                                                                                           digestion.          In water-extracted                     residue        all chemical
      16              36.3               33.1            30.1              1.20            analyses          were      made         on polysaccharides                      prepared
      30              33.8               26.6            25.3              1.33            by enzyme            digestion.           Anionic         sites     were estimated
      36              33.1               30.9            23.2              1.43            from       the hexuronic              acid     and sulfur            content.

                                                                                                                         Exp                                                               onic
                                                                                                                                                                                           Ani            (I)
                                                                                                                                     Bnd          e    osam-
                                                                                                                                                       Hex-        ronic
                                                                                                                                                                  Hexu-       Sulfur
                                                                                                                                              )          inc        Acid                   Sites      71)
   3.     Dyebinding                   and           mucopolysaccharide                                                                                                                      (2)
content             in    cartilage             : The       amount           of    dye      bound
                                                                                                                                                                                          micro-
by cartilage           is ten times            as much         as that        bound         by                                                                                            eyusva-
                                                                                                                                     gz,noles         motes       gsmoles     psnotes
the mucoid           layer       of the comb.            The water-extracted
tissue      binds      approximately                 half the amount                        In
                                                                                     of the tissue                         31          409              193         167           166       333      1.22
unextracted             tissue.       The       dyebinding             of the         water                                34          420                -         147           147       293      1.43
extract        accounts          for the difference                 (Table        VI).                                     45          421
    The     number          of anionic          sites     calculated          from       the
chondroitin            sulfuric         acid      content          of the       tissue      Inis the                       31          187                70         79            62       141      1.32
approximately                30%       lower        than      the     moles         of dye water                           34          171                46         50            53       103      1.66
bound        by the         tissue,        suggesting           that       in addition          extract                    45          162                41         35            -         -        -


to the        anionic         sites      of chondroitin               sulfuric         acid, of! gram
there      are other         sites    present          in the cartilage              which of tissue
bind       the cationic                dye.        In the water               extract           and       in
the       water-extracted                  residue           the      molar            ratio         of Inthe                    31      209         89        61        61       122    1.77
bound          dye-anionic                sites      is also more              than        one.              water               34      264         78        89        81       170 1.55
     A further              study            was       made        on the             correlation            extracted           45      250
between            the      amount              of dye         bound          and        the hexu- residue
ronic        acid      content              of the        extract.           This         was       done of 1 gram
by extracting                  various            amounts           of cartilage                 tissue of tissue
in the same              volume             of distilled           water.          The       hexosa-
mine,         hexuronic            acid         and nitrogen                contents            of the  extract        the ratio           of the dyebinding                     at pH 6.1 and
crude         extract         were         determined.              The         total        amount
                                                                                                        2.05 is 2.2.
of polysaccharides                        and proteins               in the various                    ex-
tracts          increases             as the            amount            of tissue               is in-
                                                                                                                                          DISCUSSION
creased           (Table         VII).           The      ratio        of the bound                   dye
to the anionic                 sites        is between 1.54 and               1.74 in the                    The      correlation              between            the      dyebinding              and
various extracts.                  The protein-mucopolysaccharide                                       the        mucopolysaccharide                    content             of      a     tissue
ratio       is constant              in the different                   extracts.                       hinges       on        the      reliability            of     the        mucopolysac-
    Water           extract            of cartilage                was        digested              withcharide           determination               method.             Direct         hexos-
testis       hyaluronidase                    and dialyzed               against           distilled
                                                                                                        amine        or hexuronic                 acid      determination                made        on
water         to remove               the buffer             and      the small              molecu-
                                                                                                        tissue        slices        does         not       give       a measure                of    the
lar      degradation               products              of the          chondroitin                  sul-
                                                                                                        mucopolysaccharide                       content            because             of       the
furic        acid.       The         hexuronic                acid       and         hexosamine
                                                                                      undigested presence
                                                                                                                            of      hexosamine-containing                   macromole-
contents            dropped              to one-sixth               of the
extract.          No measurable                      amount          of dye was bound                   cules         other            than            mucopolysaccharides                and
by the           extract          after          hyaluronidase                   treatment              because
                                                                                                          at             the known               hexuronic           acid       determination
pH 6.1 and 2.05                      (Table           VIII).         In the           undigested methods                  give      erroneous            results        in the presence               of




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422                                                                     ENDRE                        A. BALAZS                            AND            JOHN              A.         SZIRMAI



                                                TABLE                      VII                                                               This         indicates                    that           large          molecular                      nondialyzable
Dyebinding                        and      Mucopolysaccharide                                          Content                       of      mucopolysaccharides                                       are           not              removed                      during
                                     Cartilage      Extracts                                                                                 proteolysis.                       However,                       small           molecular                     dialyzable
    Hexuronic             acid     was     determined              on
                                                                  dialyzed         mucopolysaccharide                 components          present                                                                                                           in the
extracts.         Anionic         sites    were       calculated          as 2 tissueX         will be removed            in the course            of prepa-
                                                                                                                                                  this
micromoles           of hexuronic           acid.     The       protein       con- ration      method.
tent      of the lyophilized            extracts was calculated              by        With       distilled       water      extraction,          approximately
multiplying             the    total     nitrogen          content         by    6.25 % of the hyaluronic
                                                                                   90                                       acid      can be removed              from
after       subtraction             of the        hexosamine            nitrogen. the     mucoid            layer    of the        comb      of adult          roosters
                                                 Water           Extract            of     1              Lyophilized                        and          50%              of         the           chondroitin                        sulfuric               acid                 from
               Amount    of                            gram        of Tissue                          Water            Extract
                  Tissue
                                                                                                                                             the         cartilage.                     This            effective                    extraction                      of            both
Exp.         * Extracted                                                   Ani-                                                              mucopolysaccharides                                        with              distified                   water                        is
                in 200 ml               Bound           Hexu-                                                            Pro-
                                                                           onic           (1)        Hexos-
                   Water                  dye            ronic
                                                                           sites                      amine               tein               probably                      due            to         the           fact           that              sectioning                           the
                                           (1)            acid               (2)
                                                                                                                                             tissue    with                     the      freezing       microtome          gives                                         a        good
                                                                       micro-                                                                homogenate.                               The        aqueous         extracts                                          of             both
                         mg             pmoles           pmoles         equiv-                              %                %
                                                                        otenis                                                               tissues         after               dialysis               contained                      approximately                                    40
15-B                 119.1                 166   47.7                      95.4 1.74 11.5                                  37.0              to      50%           proteins.
15-C                 278.2                 160   52.2                      104.41.53 13.3                                33.0                       The           chemical                     analysis                   of         the        purified                     muco-
15-D               539.7                   130   42.1                      84.2 1.54 13.2                                 33.0               polysaccharide                               fractions                  of        the          total          tissues                  and
15-E              1182.8                    115 37.3                       74.6 1.54  15.8                                 49.0              their         water                extracts               indicates       that                          the mucopoly-
                                                                                                                                             saccharide                         present             in the       mucoid                             layer   of the
                                                                                                                                             comb            and            in        the cartilage                 of the   septum        nasi                                    is
                                               TABLE                    VIII
                                                                                                                                             predominantly                            hyaluronic                     acid  and     chrondroitin
Effect             of         Hyaluronidase                        Digestion                         on          the          Water
                                                                                                                                             sulfuric              acid,     respectively.
                                    Extract                    of Cartilage
                                                                                                                                                 When                dialyzed          aqueous                                 extracts                of          cartilage
       1.8    g     of        cartilage                   tissue              was          extracted                          with           100
                                                                                                                                             and          comb             are          added                to      dye             solutions                of         various
ml of distilled       water.                                     Hexosamine                       and     hexuronic
                                                                                                                       concentration,             one    obtains                                                               color            changes                      in         the
acid  were      determined                                          on    the                dialyzed        extract.
                                                                                                                       solution       similar         to      those                                                              obtained                     with                      pure
The  acid     dyebath        of                                  pH    2.05                was     adjusted         with
HC1.                                                                                                                   chondroitin         sulfuric       acid      or                                                         with          hyaluronic     acid.
                                                                                                                       In the latter          case,   depending                                                                 on         the mucopolysac-
                                                               Water         Extract             of 1 gram               of
                                                                           Cartilage             Tissue                                      charide-dye                   ratio            in the             solution,                   a metachromatic
  Exp.             Dial    zed                                                                                                               shift of the absorption                                         band   of the dye will                                           occur
    *          Water    xtract                                                                                    Dye
                                                      Hexu-                                   Dye
                                                                                           bound            at bound                 at
                                                       ronic           Hexos-                                                                or a precipitate      will                                    be formed.     Similarly,                                          when
                                                       acid            anune                    pH        6.1      pH         2.05
                                                                                                                                             excess              amounts                       of      dye          are          added                to       the                water
                                                  j.smoles              pmoles                  .imoles               pmoles                 extracts              of these                    tissues, a precipitate       will                                     form
45-E              Before       di-                     34.4                 40.9                  162                     72                 and      the           amount                      of dye   in the precipitate                                            will                  be
                     gestion                                                                                                                 directly               proportional           to the       mucopolysaccharide
45-D              After      di-                         6.6                  6.0                 <2                    <2                   content                of the    extract.       The     precipitate         will                                                  con-
                     gestion                                                                                                                 tain          all       of the        mucopolysaccharides           present                                                     in
                                                                                                                                             the         water         extract.
                                                                                                                                                    In    the        water      extract                           of the             mucoid                layer             of the
other               carbohydrate                          components                                       of           tissues.
                                                                                                                                             comb        of adult    roosters        and     testosterone-treated
Dialyzed                distilled                       water     extracts                                  of         comb                and
                                                                                                                                             cockerels,        the molar       ratio    between         the hexuronic
cartilage                     slices,             however,                         give               hexuronic                           acid
                                                                                                                                             acid     and    the bound        dye    is close     to one.         This   sug-
values              identical                    to        those                 obtained                        on       the             pure
                                                                                                                                             gests      that     the contribution            of substances             other
mucopolysaccharide                                    fraction.
       The         hyaluronic                         acid          and             chrondroitin                           sulfuric than         hyaluronic           acid     to the     binding          of cationic

acid         contents                     of     comb       and
                                                              cartilage were                                            deter- dye            is negligible.            In cockerels,         the     daily      dose     of
mined          after              the          quantitative        preparation                                            of these  testosterone            increases        the   comb        weight        and      the
mucopolysaccharides.                                      The    pepsin-trypsin                                           diges- total amount                  of hyaluronic         acid    in the comb            (18).
tion   procedure                               used        to remove         the                                proteins                      Similarly,
                                                                                                                                           pro-                            the         amount                   of dye                bound            by          the             total
duces               no           dialyzable                          breakdown                                    products                    comb
                                                                                                                                                of               tissue,               by           itswater           extract                  and           by             the
hyaluronic                       acid     and                     chrondroitin                                  sulfuric                  acid.
                                                                                                                                              extracted                     tissue                  residue                also              increases                       during




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                        DYEBINDING                                   AND            MUCOPOLYSACCHARIDE                                                       CONTENT                 IN         CONNECTIVE                          TISSUE                           423


treatment.                          This             correlation                            between                          the             hyalu- of the         comb            the        amount                  of hyaluronic                           acid         can      be
ronic           acid           content                    of the              tissue             and          the            amount                   of
                                                                                                                                                    determined                  by         the dyebinding          method.
dye           bound                     by      the    tissue      in                            the      course       of                           tes- Dyebinding                       does   not     provide       a quantitative
tosterone-induced                                 comb        growth                                   is    a    further                           determination                         of the   free    anionic      sites  of muco-
indication                       that          most    of the dye                                  is taken      up by                              the
                                                                                                                                                     polysaccharides                     because                the         total              amount                of     dye
anionic                sites            of         the          hyaluronic                       acid.                                              bound           by       the          tissue            is,        as        a rule,               more           than          the
      In      the           mucoid                  layer               and          in the              water-extracted                            total       number                   of    anionic                   sites           calculated                   from           its
tissue    the molar   ratio     of bound      dye-hexuronic                                                                                   acidmucopolysaccharide                                 content.                       It      does,    however,
is higher     than  one.    This    indicates       that    the                                                                           contri- give    a quantitative                                  account                         of certain       anionic
bution                 of          proteins,                         nucleic                acids,                 and                  possibly sites         of the         tissue;              viz.,           (1)      the           free     anionic                sites         of
some            other              tissue                 components,                              to        the          dyebinding                macromolecular                     components                                which,       upon                               ex-
is not              negligible                      in          these              cases.           The                 contribution                traction      into              the dyebath,                            form    a precipitate                                with
of      the         phosphate                             ester             groups                  of       nucleic    acid the  to dye      and   (2) the                                                     anionic                  sites          which              are      at-
the cationic dyebinding                                                   the
                                                                         of               tissue            is probably         tached      to components                                                             insoluble                   in        the       dyebath
not too great.  It was                                                found              that            the total       nucleicand    free      to  interact                                                      with                  the       dye              molecules.
acid          phosphorus                             in         the         cross           section                     of        the         combConnective                        tissues                 contain                       large             amounts                     of
of      adult           roosters                     is only                   1.6        micromoles                               per         grampolyacids               which      interact     with     cationic                                           dyes.         This
of wet               tissue                   due          to          the           low         cell          content                             interaction
                                                                                                                                              of the                          causes        a metachromatic                                                  shift         of the
mucoid                layer.                                                                                                                       absorption                 spectrum         of the       bound                                             dye.          While
      Using             cartilage,                        its         water              extract                   or        the          water- tissue            metachromasia                                gives             a qualitative                           picture
extracted                        tissue,                 the           molar                ratio             of         bound                   dye- the
                                                                                                                                                    of        distribution                                 of      the      polyacids          interacting
anionic                sites,                calculated                        from           the            hexuronic                              with
                                                                                                                                                 acid     the     cationic                         dye            molecules,          dyebinding           is a
and         the         sulfur                content                   of the              mucopolysaccharide                                      quantitative                         measure                      of         the             amount                   of        dye
fraction,                   was              found                to        be          higher               than                 one.
                                                                                                                                  This              molecules                bound                   by            the            anionic                   sites          in       the
suggests                    that             the          contribution                           of binding                              sites        of
                                                                                                                                                    tissue       (21).
tissue                 components                                      other                than                   mucopolysac-
                                                                                                                                                                                                SUMMARY
charides    is not                             negligible.                      Approximately                                 85%       of
the chondroitin                                 sulfuric                     acid    in the                        water       extract     The     hyaluronic          acid      content           of the       mucoid
of      cartilage                   is removable                                   by       dialysis                 after      hyalu- layer    of the        rooster       comb         and     the     chondroitin
ronidase                    digestion                       and               no         precipitate                       is formed sulfuric        acid      content        of
                                                                                                                                                                              bovine         nasal   cartilage
following                the            addition                      of dye.                                                          were    determined           in the     freshtissue,    in the water

      The            amount                         of          dye               cartilage extract
                                                                               bound                    by  of the the    tissue,        and      in the        water-extracted
                                                                                             residue.         The         binding            of     cationic          dye        by    the
extract         in acid        (pH      2.02)       solutions            was less than
                                                                                             tissue,       the       tissue       extract           and       the      residue        was
half     the amount              bound         in aqueous              (pH 6.1)          solu-
                                                                                             compared             with       the    mucopolysaccharide               content.
tions.      This        indicates           that      half       of the         total        dye
                                                                                             The      molar        ratio       of the        bound        dye     to the anionic
bound         interacts          with      anionic         sites      still   ionized          at
                                                                                              sites    of the        mucopolysaccharides                 varied        between
pH      2.02, very probably the ester                         sulfate       group         of
                                                                                              1.1 and         1.8.      In the        comb         extract        this      ratio     was
chondroitin             sulfuric        acid.      Proteins           present          in the
                                                                                             one.     This      indicates         that,       with      the exception             of the
tissue       sections         or in the           extracts          do not         seem comb    to      extract,         anionic        sites    other       than      those      of the
block       the      anionic         groups         of chondroitin                  sulfuric mucopolysaccharides                   are also          responsible            for the
acid,           leaving                   them                 free          to         interact                   with                the       dye.
                                                                                                                                                   dyebinding.
If, in          oivo, proteins                            occupy                   the       anionic                    sites            of the
                                                                                                                                                                                     ACKNOWLEDGMENTS
mucopolysaccharides,                                                  the               conditions                                of          the
dyebinding                           experiments                                  are        such              that                    they         are The            assistance                  of           Misses                   Doris              M.        Bunney,
replaced                    by          the         dye           molecules.                                                                        Marcella               H.        DeRoche                          and           Alice              F.        Feldman                 is
      The            determination                                      of          dyebinding                               in          general gratefully                 acknowledged.
does          not            give             a quantitative                                 account                         of         the      acid                                     REFERENCES
mucopolysaccharides                                             in          the           tissue.                   Combining
                                                                                 1. ANDERSEN,                                                                                            L. A spectrophotometric            method
dyebinding        with      hyaluronidase               treatment           does       for     the                                                                                      determ1nation            of     sulphate         and
not necessarily        make       this    determination             possible.          organic                                                                                       sulphur        on the       micro-       and   ultra-
                                                                                       micro-scale.                                                                                          Acta     chem.     Scand.       7: 689-692,
However,        in water       extracts        of the        mucoid        layer       1953.




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424                                                 ENDRE           A.      BALAZS               AND      JOHN          A.     SZIRMAI


  2. BALAZS,           E. A. Unpublished             data.                                                 the       mechanism                involved.           J.       Biol.      Chem.
  3. BALAzS,            E.     A. AND      ALLGOTH,       A.-M.                     Dye-                   184:      321-332,         1949.
        binding            and    mucopolysaccharide         content                    of        14.   IMMERS,         J.   AND       VASSEUR,         E.    Interaction            of
          connective                tissues    of    the     eye:        cornea,       sclera,             amino       compounds                 with      monosaccharides,        a
          vitreous            body.    (In preparation)                                                    s_pectrophotometric                 study      of the colour          with
  4.   BALAzS,           E.     A. AND     BERGENDAHL,                      G.      Dye-                   Ehrlich’s          reagent.             Acta       chem.         Seand.             6:
          binding             of the      developing                     rat       skin.         (In       363-375,      1952.
          preparation)                                                                            15.   KLOTz,       I. M.       Protein        interactions.          Chapter       in:
  5.   BALAzS,           E.    A.      AND    MAYER,          N.      H.      Quanti-                       The Proteins.            Chemistry,              Biological          Activity,
          tative          aspects        of    the         interaction            between                  and Methods,           Vol.       I, Part           B, edited            by H.
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