Changes in Side Chain Reactivity Accompanying the Binding of

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       Vol. 239, No. 2, February     1964
             Printed   in U.S. A.

      Changes                 in Side Chain Reactivity Accompanying                                                                    the Binding
                                of Heme to Sperm Whale Apomyoglobin*
                                                                          ESTHER     BRESLOW

                 From     the Department           of Biochemistry,     Cornell   University      Medical     Co&e,      New    York   21, New      York

                                                              (Received for publication, August 30, 1963)

      Considerable attention has been devoted recently to the study                       present was oxidized by either CuCIZ (9) or K3Fe(CN),r followed
  of conformational      changes occurring during enzyme-substrate                        by exhaustive dialysis finally against distilled HzO.
 interaction     (1, 2). Presumably the nature of the interaction                            Initially globin was prepared from metMb2 by acetone precipi-
 between apoproteins and their prosthetic groups is essentially                           tation according to the method of Theorell and ikeson (10).
  similar to that between enzymes and substrates, with the excep-                        Subsequently,      a modification      of Teale’s 2-butanone extraction
  tion that no catalytic step occurs subsequent to the initial bind-                     procedure (11) was employed.            An approximately        1 y0 deionized
 ing. Studies of the physicochemical           changes accompanying the                  solution of metMb was lowered to pH 1.5 at, 0” and then ex-
 binding of heme to sperm whale apomyoglobin             would seem to be                tracted at 4”, first with an equal volume of 2-butanone (Merck

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 of particular interest in t.hat the three-dimensional        conformation               reagent) and twice more with half-volumes                 of 2-butanone, al-
 of sperm whale metmyoglobin          is now known (3), and conforma-                    though the last extraction was generally found to be superfluous.
 tional changes occurring on heme-globin interaction may there-                          The resulting, slightly straw-colored         solution was then immedi-
 fore be more amenable to interpretation.                                                ately dialyzed at 4” in 18/32 Visking tubing, usually first against
      Kinetic studies of heme-globin        interaction  suggest that the                several changes of dilute NaHC03 (50 mg per liter) and finally
 initial product of the reaction must rearrange to form the native                       against several changes of glass-distilled            HzO. The resultant
 heme-protein       (4). Conceivably this rearrangement          involves a              solution was concentrated to less than half it,s original volume
 change in protein conformation.             Optical rotatory     dispersion             by evaporation through the dialysis membrane, and then cen-
 studies of metmyoglobin        give values for the a-helix content of                   trifuged to remove some colored precipitate, t,he protein solution
 approximately        75% (5, 6), whereas similar studies of globin                      itself being completely        colorless in good prepa.rations.            This
 suggest a helix content of 50%.’         In view of the differing Cotton                solution was then adjusted to be 0.16 M in KCl, and any addi-
 effects in the two proteins, however, it is difficult to assess the                     tional precipitate      was removed by centrifugation.                (It was
 significance of this disparity.       In a preliminary    communication                 found, however, that only when the original NaHC03 dialysis
  (7), this laboratory     cited discrepancies between the H+ ion                        was omitted was any additional protein precipitated                by KCl, so
 titration curves of globin and metmyoglobin             which could pos-                that this step is unnecessary if NaHC03 dialysis is introduced.)
 sibly be interpreted       as indications      of subtle conformational                 Many properties of the globin so obtained have already been
 differences.     The primary aim of the present study is to examine                     described (7). Two additional observations are also of interest.
 in greater detail the significance of the relative reactivities of                      First, removal of the heme is completely insured only if the pH
globin and myoglobin derivatives to H+ ion. Secondarily the                              of the initial metMb solution is near 1.5. Second, the stability
 observed differences in reactivity of imidazoles to H+ ion in the                       of the resultant globin is markedly lowered in the presence of
 two proteins will be related to changes in reactivity to bromo-                         heavy metal ions. In the cold, in 0.16 M KCl, the presence of
acetic acid, a reagent for which the reaction with metmyoglobin                          trace metal ions will result in the gradual precipitation                of de-
imidazoles has been recently documented (8).                                             natured globin.       In recent preparations,       inclusion of a dialysis
                                                                                         step with 1 X lop4 M disodium EDTA before dialysis against
                          EXPERIMENTAL            PROCEDURE                              redistilled Hz0 resulted in globin preparations             which were per-
                                                                                         fectly stable for over 1 month when stored in 0.16 M KC1 at 4”.
   Reagents-Sperm      whale myoglobin was generously supplied by                        Apart from stability on standing, however, no differences among
Professor F. R. N. Gurd.         All reagents, unless otherwise speci-                   all the globin preparations in properties such as [ar]:‘, sedimenta-
fied, were analytical grade, and water was glass-distilled.                              tion constant, or titration        were apparent, irrespective of the
   Preparation   of Globin-Globin       was prepared only from met-                      method of preparation.
myoglobin.     To insure that myoglobin was ent,irely in the Fe3+                            Globin solutions were standardized            either directly by dry
state before removal of heme, all myoglobin preparations         were                    weight determinations        or indirectly     by the Folin-Lowry           (12)
examined spectrophotometrically,           and any reduced protein                       assay, with a standard curve obtained with known weights of
   * This investigation    was supported by Grant HE-02739 from                              Combination     of Globin with Heme-The              heme preparations
the National Heart Institute, United States Public Health Serv-                          used for recombination        studies were either hematin, C grade
ice. A preliminary      account of this work was presented at the
145th National Meeting of the American Chemical Society, Sep-                            (Calbiochem),       or analyzed hemin, 8.61% iron (British Drug
tember 9 to 13, 1963, New York.                                                             2 The abbreviations used are: m&Mb, sperm whale metmyo-
   1 J. Schellman, personal communication.                                               globin; Mb, myoglobin; metHb, methemoglobin; Hb, hemoglobin.

February      1964                                                       I:‘. Breslow                                                                         487

Houses, Ltd., London).3             The combining capacity of globin for          central thermostated          bath w-as pumped in parallel through the
heme was determined as follows.              A small quantity of heme was         thermospacers of the Beckman DU spectrophotometer                       and the
dissolved in 0.05 ml of N NaOH and immediately                       diluted in   water-jacket.ed,       covered titration      vessel in which the optical
borate buffer, pH 9.2, to a final concentration of 1 mM. Increas-                 density and pH, respectively,              were simultaneously        recorded.
ing aliquots of the heme solution were then immediately added                     The Radiometer TTTla             pH meter was used for all studies at 25”
to globin solutions in borate buffer, pH 9.2, ionic strength 0.16.                and 41”, and the Radiometer model 4 was used for studies at 9”.
At varying time intervals, aliquots were taken and diluted in                     pH standards used were 0.05 M potassium hydrogen phthalate,
FDTA-acetat’e        buffer, pH 5.6 (13), and the optical density at              Beckman pH 7 and pH 10 standards, and 0.01 M NaOH in 0.14
409 rnp was compared wit.h a similar series of solutions containing               M KC1 with        Harned activity coefficients (16). For studies at
heme but no globin.           With this method, combination was found             25” and 41”, protein solutions were prepared at room temperature
to be almost complete within 5 minutes and completed within                       before optical density and pH recording.                   For studies at 9”,
2$ hours. The combining capacity so determined was 1 mole of                      solutions were prepared at 0” and then equilibrated                    at 9” to
hcme per mole of globin, although spurious results were obtained                  minimize      irreversible     temperature      effects (although      this has
if the heme solution was allowed to remain in alkali too long                     subsequently been found unnecessary).                At 41”, a faint turbidity
before addition to globin.                                                        was present in globin solutions near neutrality                   at 0.16 ionic
    Isolation of regenerated metMb apparently free of either excess               strength, but not at 0.02 ionic strength.                Values of percentage
globin or heme was accomplished on a large scale by mixing a                      of ionization       at this temperature      were therefore calculated by
 1 yh solution of globin with a slight excess of hemin in borate                  using the molar extinction at pH 7 found for the lower ionic
buffer, pH 8.9, ionic strength 0.16. The resultant solution was                   strength.
exhaustively       dialyzed against HzO, and a slight precipit,ate                    All optical densities were determined in l-cm covered cuvettes.

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 (presumably       containing      excess heme and some protein) was              Temperature         regulation was within +O.l” at’ 25” and 41”, and
removed by centrifugation.             Dry weight, spectral, and titration        within +0.5” at 9”.
studies of the resulting supernatant             revealed the product to be           Optical Rotation Studies-Optical             rotation studies were per-
essentially indistinguishable            from native metMb, as will be             formed at the sodium D-line with a Schmidt and Haensch polar-
discussed subsequently.            Recovery of metMb by this met,hod              imeter, model 58, in a room regulated at 19”. Studies of the
was 82 %.                                                                         effect of pH on rotation of globin at different temperatures were
    Ultracentrifugal      Xt&ies---Sedimentation          rates of globin and      performed as follows.          An approximately        1% solution of globin
metmyoglobin         were determined in a Spinco model E ultracen-                in 0.16 M KC1 was adjusted with N NaOH to the desired pH at
trifuge equipped with phase plate schlieren optics and automatic                  the appropriate          temperature.      The solution was then trans-
temperature       control.     With Spectroscopic II-G plates (Kodak)             ferred to the covered polarimeter tube which was immersed in a
for metMb studies, usable patterns could be obtained without                      water bath at the same temperature.                The tube was maintained
the use of special filters at concentrations           near 1% by increasing      in the water bath until immediately before the readings, at which
the exposure time to 7 seconds.                                                   time it was quickly dried and readings were taken over a period
    Hydrogen Ion Equilibria-The               isoionic pH was determined          of about 5 minutes and extrapolated to zero t,ime. The temper-
after passage through a Dintzis deionizing column (14). Con-                      ature of each solution can then probably be regarded as accurate
tinuous potentiometric            H+ ion titrations        were performed as      to only within ~2”.
previously described (13). For globin and metMb, continuous                           Reaction of Globin. with Brornoacetic Acid-Bromoacetic                  acid
titration curves were obtained between pH 11 and 3 at 25” and                      (Fisher, “highest purity”)          was recrystallized      from toluene and
41” and between pH 12 and 3 at 9”. Several additional poinm                       petroleum ether.
at more alkaline pH were obt,ained by discontinuous titration.                        To native globin in 0.16 M KCl, pH 7, enough of a self-buffered
    Spectrophotometric         studies of phenolic equilibria       were made     solution (pH 7.9) of K2HP04 and bromoacetic acid was added so
with globin, metMb, CN--metMb,                    and CO-myoglobin.         CO-   that the final conditions were 1% globin, 1 M phosphate, and
Mb for this purpose was prepared as described by Hermans (15))                    0.2 M bromoacetic acid, pH 7.4. The mixture was allowed to
stored under CO, and standardized by dry weight determination.                    stand at room temperature with occasional shaking for 7 days.
C--m&Mb           studies were conducted with metMb in the presence               Moderate precipitation          was generally observed during the course
of 0.01 M NaCN; the cyanide derivative was spect.rophotometri-                    of the reaction.           In one reaction, therefore, precipitate           and
tally demonstrated         to be stable to pH 12.9 at 25”.                        supernatant        fractions were separated and then individually
    In globin, studies of tyrosine ionization were conducted at both              exhaustively dialyzed against H20 and lyophilized.                  Amino acid
245 and 295 rnp with essentially identical titration curves calcula-              analyses of the two fractions? indicated no significant differences.
ble at both wave lengths.             As previously cited (7), globin tyro-       In the subsequent reaction, then, precipitate and supernatant
sine ionization studies at 0.16 ionic strength gave identical results             were not separated but dialyzed, lyophilized,                    and analyzed
whether conducted at 0.05% protein concentration                    in glycine-   together.      Amino acid analyses of the two runs gave identical
 KC1 buffer or at 0.4% protein concentration                in KC1 alone. All     results.     Tryptic hydrolysis and peptide mapping of the car-
studies reported here, therefore, were conducted at approximately                 boxymethylated         globin preparations were performed by Dr. L. J.
O.OSy, protein concentration            in 0.01 M glycine-KC1 buffer, 0.16        Banaszak and Professor F. R. N. Gurd as previously described
ionic strength.         The optical densities of all protein solutions             (8).
were obtained versus Hz0 ait.h the appropriate                   buffer blanks        One reaction with acid-denatured           globin was also investigated.
subsequently subtracted.                                                          Here, globin in 0.16 M KC1 was first allowed to stand at pH 2.9
    To insure adequate temperature                regulation,   water from a         4 Amino    acid analysis   and peptide     mapping   of carboxymethyl-
                                                                                  ated globin    were performed    by Professor    F. R. N. Gurd and Dr.       L.
   3 This   hemin    was   a gift   of Dr.   Nevenka   Rumen.                     Banaszak.
488                                                               Changes Accompanying                                   Heme Binding        to Apomyoglobin                                     Vol. 239, No. 2

                                                  TABLE           I                                                            native metMb and in regenerated                       metMb prepared from 2-
 Major         absorption       bands of nativs                  and      regenerated metmyoglobin                             butanone-extracted               globin are compared.        It is readily apparent
                                                                                                                               that no significant difference exists between the two proteins.
                                                         T                             enBY
                                                                                        -                                      In Fig. 1, the titration               curve of the same regenerated metMb
      Sative                    Regenerated                            Native                      Regenerated
                                                                                                                               is compared with that obtained for native metMb.                          No signifi-
                                                                                                                               cant differences between the two proteins are apparent upon
          my                           WJ                               X104                           Xl@                     titration from pH 9 to 3 with HCl.                     The back-titrat.ion     curve,
         630                        630-635                             0.35                           0.35                    obtained by rapid titration from pH 3 with NaOH, is similar to
         505                           505                              0.93                           0.91                    that obtained with some but not all metMb preparations.                          The
         409                           409                             16.0                           15.9                     reason for this behavioral                  difference among various metMb
         280                           280                              3.1                            3.2
                      I                                                                   I                                    preparations is not apparent, but as the continuous rapid back-
                                                                                                                               titration        represents a nonequilibrium             situation during which
                            I                 1              I                  I              I                     I         some native protein appears to be regenerated, it may be simply

                                                  REGENERATED                            NATIVE                               a reflection of differences in protein concentration or in the length
      30                                                                                                                      of time for which t,he protein was allowed to remain at pH 3.
           i                    I     pH9+-3              0.0                             -                          1
                                                                                                                              In any event, the globin studied appears to have a conformation
                                      pH3+8              000                             ---                                  capable of reacting with heme to give metMb.                         Presumably the
                                                                                    (2 different
                                                                                     preparations)                            preparation            of globin by the 2-butanone extraction procedure,
                                                                                                                              which involves exposure to pH 1.5, is not deleterious despite the
                                                                                                                              fact that globin is denatured at this pH (7), because this de-

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      20                                                                                                                      naturation, like that of metMb itself, is reversible.
                                                                                                                                    Ultracentrifugal          Studies of Globin and Metmyoglobin-A            some-
                                                                                                                              what crude indication of the relative conformations of globin and
      15 c                                         PRECI          PlTATlON                OF                                  metMb may be obtained from the ratio of their frictional coef-
                                                                  DENATURED                        FORM
                                                                                                                              ficients     (.fglobin/fmetMb).          F rom t,he known molecular         weight of
                                                                                                                              the two proteins and their sedimentation                  rates at infinite dilution
                                                                                                                              under comparable                 conditions      of pH and ionic strength, this
                                                                                                                              ratio may be obtained from the equation

                                                                                                                                          f- globin     =   S2o,w(metMb) mol. wt.,l,bi,(=17,197)
                                                                                                                                                                          X                                     (1)
                                                                                                                                          fmetMb            &Xl,w(globin)   mol. wt. metMb(=     17,830)

                                                                                                                              The sedimentation      rates of globin and metMb at several con-
                                                                                                                              centrations in phosphate-KC1           buffer, ionic strength 0.17, pH
                                                                                                                              6.74, were determined        at 25” and, although unnecessary for
                                                                                                                              comparative purposes, were corrected to ~20,~ by using values of
                                                                                                                              0.74 for the partial specific volume and density and viscosity
                                                                                                                              data interpolated    from the International         Critical Tables.       The
                                                                                                                              results are shown in Fig. 2. For metMb, .sz~.~ at infinite dilution
                                                                                                                              is 1.98. For globin, the best value of .sz~,~ at infinite dilution is
                            4                 5
                                                            6~H7                               8                 '            1.87. The measured ratio of frictional coefficients, 1.02, would
  FIG.      Experimental
           1.             titration   curves of native metMb and                                                              seem to suggest that no major conformational                difference exists
metMb regenerated from globin and heme. Temperature,           25”;                                                           between the two proteins.            Theorell    and ikeson         (10) have
ionic strength, 0.16. For regenerated metMb: l , titration from                                                               reported values of szo,W at infinite dilution of 1.84 and 1.96 for
pH 9 to pH 3; 0, back-titration     from pH 3. For native metMb:                                                              horse apomyoglobin       and CO-myoglobin,          respectively.       On the
--, titration from pH 9 to pH 3; - - -, back-titration  of two dif-                                                           assumption that the horse and whale myoglobins have the same
ferent preparations from pH 3.
                                                                                                                              molecular weights, these sedimentation             data similarly suggest
                                                                                                                              that no definitive conformational        differences between globin and
for 30 minutes at room temperature and then rapidly adjusted
                                                                                                                              myoglobin are observable by such gross measurements.                       Vis-
to pH 7 with NaOH before addition of KtHP04 and bromoacetic
                                                                                                                              cosity studies by Eylar5 of sperm whale globin and metMb lead
                                                                                                                              to similar conclusions.
                                    RESULTS        AND       DISCUSSION                                                          Equilibria  of Imidazoles with H+ Ions in Globin and in Met-
                                                                                                                              myoglobilz-Analysis      of the titration curve of metMb has shown
   Properties of Regenerated Jfetmyoglohin-The    ease of regenerat-
                                                                                                                              that only 6 of the 12 metMb imidazoles are in H+ equilibrium
ing native metMb from globin prepared by acid-acetone pre-                                                                    in the native protein and that the remainder were masked in the
cipitation has been well documented       (10). On the other hand,
                                                                                                                              unprotonated     form and released upon acid denaturation                 (13).
globin prepared by 2-butanone extraction has not been as thor-
                                                                                                                              In a preliminary      account (7), differences in H+ ion titration
oughly studied.      As previously     stated (see “Experimental                                                              between globin and metMb in the neutral pH range were inter-
Procedure”),    we have found no demonstrable     differences in the
                                                                                                                              preted to indicate the release of 2 to 3 additional imidazoles into
globin prepared by these two procedures provided that all ma-                                                                 H+ ion equilibrium       upon removal of heme from metMb.                    In
terial insoluble in 0.16 M KC1 at pH 7 to 8 is removed.      In Table
I, the positions and intensities of the major absorption     bands in                                                            5 E. Eylar,          personal   communication.
February        1964                                                                  E. Rreslow                                                                                                                                            489

some small part, this analysis was based on a tentatively revised
 (and now abandoned)         assumption of a total of 11 histidine
residues in metMb.6       The following more detailed accounting of
globin imidaaole H+ ion equilibria          will rest on the now more
definite total of 12 metMb imidazoles.              It should be noted,
 however, that there is a discrepancy between the observed
isoionic pH values of metMb and globin and the most recent
amino acid analyses (17), some small part of which may reside
in the imidazole analysis.
    In Table II, the current estimate of titratable groups in metMb
is given. The isoionic pH of metMb is 7.86 and is unchanged by
essentially    complete guanidination        (18). No lysine residues
 therefore titrate below pH 7.86, and even if complete depro-
 tonation of imidazoles is assumed at this pH (an assumption not
completely warranted by the estimated imidazole log k’),7 it is
apparent that at pH 7.86 the calculated charge should be at
least 1.5. In globin this discrepancy is again seen. The iso-                                               l .80       ’                 I                    I             I       I                     I                  I
                                                                                                                                                                                     I                     I                  I                   I
ionic pH of 8.63 is compatible with that of metMb when the                                                                               .2           .4                   .6       .8                    1.0                1.2                I.4
effect of heme removal is allowed for (7). Here, even generously                                                                                   PROTEIN                   CONCENTRATION                            (%).
allowing for complete deprotonation           of the carboxyls, or-NHz,                              FIG.        2. Sedimentation                          of globin              and        metMb            as a function                      of

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 12 imidazoles, and 0 to 1 e-NH2 group at this pH (a number in                                  protein concentration  in potassium                                                    phosphate-KC1                    buffer, ionic
accord with the maximal acid-binding           capacity of 34 to 35 (7)),                       strength 0.17, pH 6.74. l , metMb;                                                     0, globin.
the calculated charge is 1 to 2 at pH 8.63. At present, however,
there is no recourse other than to assume the correctness of the                                                                                                   TABLE          II
amino acid analysis as given in Table II.                                                                        Tit&able                side       chains          0.i sperm - whale                  metmyo            obin
    The difference in titration      behavior of the native and acid-                                                                                                                      Log k’ in native
                                                                                                                         Side chain                                        No.              protein at        25’              Reference
denatured forms of met,Mb, i.e. the titration difference curve, is
                                                                                                                                                                     -                                                                           -
 primarily a reflection of the difference in w, the electrostatic work
factor,7 and the number of imidazoles in H+ ion equilibria in the                               or-Carboxyl*.                   ................                             1                                                  (13,       7)
                                                                                                Porphyrin             carboxyls*.                    .......                 2                         4.48                     (13,       7)
two species (13). As a first approximation,            the value of w for
                                                                                                P,y-Carboxyls*.                     .............                           20                         4.48                     (13,       7)
a given class of groups in both native metMb and native globin                                  Reactive          imidazoles.                     ........                   6                         6.70                     (13,       7)
may be considered the same,8 as the sedimentation             data indicate                     Masked         imidazoles.                     .........                     6                                                  (13)
no major conformational         differences between the two native                              a-NH2      ......................                                             1                      7.80                       (13)
 proteins.    Moreover,    w for the two acid-denatured             proteins                    Fe(OH)z+.             ..................                                     1                       8.90                       (13)
appears to be the same. Analysis of carboxyl ionization                     in                  E-NH*.      ......................                                          19                  -10.5                           (13)
denatured globin at 0.16 ionic strength and 25” can be shown to                                 Guanidinium                     ................                             4
give average values of 0.033 and 4.41 for w and log k’, respec-
                                                                                                   * Log k’ for carboxyl                               titration            was determined                     by assuming                      the
tively, for the 21 globin carboxyls, in good agreement with the
                                                                                                same acidity    for all                            carboxyl                groups.              Undoubtedly,                     however,
values of 0.034 and 4.49 obtained for the 23 carboxyls of de-
                                                                                                the a-carboxyl     log k’                             is lower             than         t,hat     of the            ot.her       carboxyl
natured metMb (13). A rough approximation                   of the relative                     groups.
number of imidazoles released into H+ ion equilibrium              in globin
and in metMb can then be obtained from the relative magnitudes                                   near pH 4.6 for metMb.          In Table III, the relative heights of
of t,he difference curves alkaline to the pH of denaturation.              At
                                                                                                 the difference curves (AT*) in the two proteins are shown.              If
25” and 0.16 ionic strength, estimat’ion of t’he difference curve is                             a release of 6 imidazoles upon acid denaturation             in metMb is
complicated by precipitation      of the denatured protein near pH 6.                            assumed, the number of imidazoles            released in globin upon
The pH of precipitation,      however, is increased to above 7 if the
                                                                                                 denaturation     most closely approximates         3. The number of
 t.emperature is lowered to 9”, and more reproducible             difference                     imidazoles estimated by t’his method to be in H+ ion equilibrium
 curves are obtained.      In Fig. 3, the titration       curves of globin                      in native globin therefore would be 9, as compared to 6 in native
and m&Mb at 9” and 0.16 ionic strength are shown. At this
 t,emperature, globin denaturation         appears to occur gradually                               A more accurate measure of the number of imidazoles in Hf
 between pH 5.5 and 5.2 as compared to a more abrupt transition                                  ion equilibrium    would depend only upon analysis of the titration
   6 F. R. N. Gurd, personal communication.                                                      curve of the native protein.         A preliminary       titration  curve
   7 The terms in Equation 2 are defined as follows:                       log Ic’ = the         analysis based upon the increased number of groups titrating in
intrinsic       H+ ion association         constant;    w = the electrostatic            work    globin relative to metMb in the neutral pH range suggested that
 factor;     s!? = the net protein        charge;    ni = the number         of titratable       8 or 9 imidazoles were in H+ ion equilibrium        in native globin (7).
 groups in a given class; and fiH = the number                  of protonated         groups
                                                                                                 This analysis rested on the assumption that the intrinsic pK of
 in that class.
     8 This assumption            is based on the more approximate              treatment        the t,itratable globin histidines was identical with that found for
 of titration       curves      with a distributed        charge model (19). It is               the same in metMb.        A more precise analysis may be obtained
 recognized,       however,        that with the fixed charge model of Tanford                   from the equation7
 and Kirkwood            (21), zu might        be expected     to vary      for different
 classes of groups          within     the same protein.                                                            pH = log k’ - wz(0.868)                                       -      log[tia/(ni            -      ;<a)]                    (2)
490                                                                               Changes Accompanying                                Heme Binding        to Apomyoglobin                     Vol.   239, No. 2

                                 I                     ,                  I                 I           I        I               ,        the alkaline branch of the globin titration curve, but does not
                          METMb                       TITRATION.                     9%.                                                  significantly affect the results.  The other log k’ values are those
                           -                           pHII--+
                           ---                                                                                                            shown in Table II.)
                                                       pH          3-8
                                                                                                                                             The data obtained suggest that the titration curves are most
                                            GLOBIN                       TITRATION.              9OC.                                     readily fit with a value of ni = 9 if a single class of histidines is
                                                  0            l           pii 11-3                                                       assumed.      The titration curves shown in Fig. 5 can be shown
                                                  0            0           pH 3-8
                                                                                                                                          to be completely reversible under the existent titration conditions
                                                                                                                                          to a value of 2 = +9.         However, significant deviations from

                                                                                                                                                                            H     pH9+3
                                                                                                                                             25                             O--O pH3+7

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      -20     !       I                                                       I                                   I               I
              3       4              5                     6              7pH               8       9            IO              II

   FIG. 3. Titration  curves of globin and met&lb at 9”, ionic                                                                                 0
strength 0.16, and partial titration curve of globin at 41”, ionic
strength 0.16. For metMb: --,       titration from pH 11.3 to pH 3;
- - -, back-titration  from pH 3. For globin at 9”: l , titration
from pH 11.4 to pH 3; 0, back-titration       from pH 3. For globin
at 41”: A, titration from pH 8 to pH II.
                                                                                                                                             FIG.  4. Continuous titration of globin at 25” in KCl. At ionic
                                                      TABLE                   III                                                         strength 0.16: l , pH 9 to pH 3; 0, back-titration       from pH 3. At
      Relative      magnitudes               o.f diference                           curves      in globin       and        in            ionic strength 0.06: n , pH 9 to pH 3; q , back-titration     from pH 3.
                  mctmyoglobin                at 9’ and                           0.16 ionic       strength
                                                                                                                                                        7.2   I     I                                           I
                                                                    AvH                                     Globin     imidazoles
            PH                                                                                                  released     by
                                         Globin                                       M&Mb

            5.8                           1.8                                              4.0                        2.7
            6.0                           1.8                                              3.6                        3.0
            6.2                           1.7                                              3.2                        3.2
            6.5                           1.4                                              2.6                        3.2
            6.8                           1.0                                              2.0                        3.0
            7.0                           0.5                                              1.6                        1.9

   * These values are based on the assumption that the number
of imidazoles released by acid denaturation in metMb is 6.
 by the usual plots of log FirI/(ni   - pin) against z, assuming vary-
ing values of ni, to determine log k’ and w for the globin histi-
dines. The average of a large number of globin titration curves
 at 0.16 and 0.06 ionic strength and 25” are shown in Fig. 4. In
                                                                                                                                                        6.0 --
 Figs. 5, 6, and 7, the aforementioned           plots are calculated from
 the data assuming values for ni of 8, 9, and 10, respectively,
 at the two ionic strengths.        (Values of ni = 7 can readily be                                                                                                I
                                                                                                                                                                    I       I
                                                                                                                                                                            I        I,       I          I
 shown to give unreasonable            results.)      ijSn was determined                                                                                     0     2       4        6        8          IO     12
 from the number of groups titrating              below the isoionic pH,                                                                                                         7
 with corrections for e-NH2, a-NHZ,                and carboxyl titration
                                                                                                                                              FIG.  5. Plots of pH + log ~&(nd - &H) versus 2 for globin
 made by using respective log k’ values of 10.30, 7.80, and 4.48.                                                                          imidazole    groups; ni = 8. l , ionic strength 0.16; 0, ionic
 (The e-NH2 log k’ was determined by independent                 analysis of                                                               strength 0.06. Temperature,   25”.
February     1964                                                       E. Breslow                                                                                        491

 linearity in Fig. 5, calculated for ni = 8, begin to occur at z =
  +7. Moreover, assuming no major change in molecular dimen-
 sions between globin and metMb, the theoretical              value of w
 calculated from approximate         H+ ion titration theory (19) has
                                                                                                                                                  ni =9
 been calculated as 0.065 to 0.069 at 0.16 ionic strength and as
 0.106 to 0.113 at 0.06 ionic strength (13). For metMb, the
 experimentally   obtained values of w at these ionic strengths were
 0.050 and 0.085, respectively.       Tanford and Kirkwood      (21) have
 pointed out that although significant deviations from the the-
 oretically derived value of ZL may be expected if the immediate
 charge environment      of each ionizing group and the number of
 groups in each class are considered, the change of w with ionic
 strength should remain relatively constant.          The value of 0.050
 for the change in w with ionic strength (Au) found for ni = 8
 seems somewhat high in view of the theoretical value of 0.042
and the experimental value of 0.035 found for metMb (13). On
 the other hand, the data shown in Fig. 6 for n = 9 form a linear
plot exactly to the pH where denaturation         begins, and then show
 the expected deviations.        Moreover, the calculated values of w
 at both ionic strengths studied are in essentially perfect agree-

                                                                                                                                                                                 Downloaded from by guest, on March 25, 2013
 ment with theory        Similar plots for values of n = 10 (Fig. 7)                                              I           I               I           I        I
                                                                                                                  I            I              I           I        I
also show the expected deviations from linearity in the known                                        0           2            4           6               8        IO       12
 region of denaturation,      and the effect of ionic strength on w is
intermediate    between that expected from theory and that found
                                                                                     FIG. 6. Plots      of pH +         log   &(ni        -        ii;~) versus 2 for globin
for metMb.      However, values of w so obtained for n = 10 seem                imidazole      groups;      ni =         9.    0, ionic           strength     0.16; 0, ionic
 very high, although they might be reconcilable with those of                   strength    0.06.      Temperature,           25”.
 metMb with the use of more rigorous titration curve treatment
    Reaction of Globin Imidazoles with Bromoacetic Acid-The                                         7.01
interpretation     of the H+ ion titration curves of metMb by ap-
 proximate titration theory, indicating the presence of 6 reactive
and 6 unreactive histidine residues, was seemingly substantiated
 by the reactivity of the protein to p-nitrophenyl             acetate (13).
 Recent’ carboxymethylation         studies of metMb (8) suggest that
8 histidines are available for reaction with bromoacetic                acid.
 Conceivably this apparent discrepancy is due to the irreversibility
of carboxymethylation,        or it might be that more rigorous treat-
 ment of the metMb titration curve would disclose the presence                                      6.2 --

of 1 or more additional reactive histidines with slightly abnormal
 log k’ values. Regardless of these difficulties, however, the
difference in relative reartivities of globin and metMb imidazoles
to bromoacetic       acid offers another convenient parameter by
 LThich subtle changes in the environment of histidine side chains
may be assessed.
    Reaction of native globin nith bromoacetic acid was carried                    FIG.     7. Plots      of pH +       log   Bin/(ni    -          FCH) versus 2 for globin
out in a manner analogous to that for metMb (8). In Table IV,                   imidazole        groups;     n; =       10.    l , ionic           strength    0.16; 0, ionic
the amino acid analyses of carboxymethylated              globin are com-       strength      0.06.      Temperature,         25”.
pared with the theoretical          amino acid composition.          Only 1
histidine residue appears to be unreactive in globin as compared                 residues were unreactive        to bromoacetic         acid. Upon acid,
with 4 unreactive histidines in metMb.              The identity of this        alkali, or heat denaturation      of metMb (8), and also upon Cu(I1)
unreactive residue was established by peptide mapping of tryp-                  denaturation,e     at least 1 methionine residue becomes reactive to
sin-digested carboxymethylated            globin as previously described        bromoacetate.        Similarly, carboxymethylation        of acid-denatured
(8). Only the FG3 histidine could be detected as a nonreactive                   globin definitively indicates an increased reactivity of methionyl
residue.                                                                         residues. It would seem, therefore, that the conformation                   of
    One possible cause of the greamr reactivity of globin than of                carboxymethylated        native globin differs significantly      from that
metMb imidazoles to bromoacetic acid could be the increased                      of the acid- or heat-denatured        protein, in which the previously
ease of globin denaturation.            The possibility   therefore exists      internal methionyl residues apparently become exposed.
that the carboxymethylated             globin analyzed was no longer                On the other hand, urea denaturation         of metMb does not seem
“native”      in conformation.      However, as shown in Table IV,               to cause a similar increase in reactivity of methionyl residues (8j,
the methionine content of native globin (as with native metMb)                   so that absence of methionine reaction may not be a necessary
was unchanged         by carboxymethylation;         i.e. the methionine         indication of native conformation.           A preliminary    investigation
492                                                                Changes          Accompanying                  Heme Binding       to Apomyoglobin                                Vol. 239, No. 2

                                                    IVTABLE                                                            directly linked t,o the heme iron and nitrogen atom 1 is hydrogen-
          dmino             acid analyses oj carbox~methylated                      native       globin*               bonded aithin the protein to a peptide bond carbonyl (3). The
                                                                                                                       change in reactivities of the C1 and EF, or EFs residues, however,
                                                                       Residues     per mole of protein
                                                                                                                       is not, readily interpretable      unless conformational          changes in the
                              Amino       acid
                                                                      Run       I                                      vicinity of these residues upon heme removal are postulated
                                                                   (supernatsnt                        Theoret-
                                                                                        Run     II
                                                                                                          ical         which render t,hem more accessible to solvent.                 In the crystal, at
                                                                                                                       least, these residues are not directly linked to the heme, nor is it
Lysine          ...................                                   16.7              17.5               19          the heme which appears to block sterically their merger with
Histidine ................                                             1.01              0.83              12          solvent .I0
Ammonia                ..                                              7.3               9.35               7             As previously cited (8), the lack of FG3 reactivity in both
Arginine.           ................                                   4.14              3.40               4          metMb and in globin is not yet interpretable                    in terms of the
-4spartic         acid.           ...........                          8.5               8.15               8          knorrn protein structure.          In metMb it is bonded to a heme
Threonine             ................                                 5.0               4.70               5          propionate side chain at its N-3 hydrogen position, but is other-
Serine              .                                                  5.6               4.57               6          wise exposed to solvent.             Although       situated in amino acid
Glutamic            acid..             .                              20.2              20.1               19
                                                                                                                       sequence between 2 lysyl residues, the t-aminos of these groups
Proline.                                                               4.3               4.06               4
Glycine.            .                                                 11.3              11.3               11
                                                                                                                       do not seem close to the histidine,lO and the expected effect of
Alanine.                                                              16.4              17.6               17          propionate interaction would be to raise log k’. A priori, there-
T:aline                                                                5.08              7.39               8          fore, this histidine would not be expected to fall into the class of
Methionine                                                             2.8               1.83               2          unprot’onated      metMb imidazoles masked to Hf ions. More-
Isoleucine.                                                            7.07              8.58               9          over, removal of heme should increase the reactivity                       of this

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Tyrosine.................                                              3.14              2.48               3          residue to bromoacetate          unless, of course, the postulated con-
Phenylalanine.                                                         6.05              6.01               6          formational       changes accompanying             this reaction place this
Leucine.                                                                                18.2               18          histidine in a more internal position.                Alternatively,     the lack
3Xarboxymethylhistidine                                ......           1.62             2.04               0          of FGs reactivity t,o bromoacet,ate, at least, may be somehow
Dicarboxymethylhistidine                               ......           9.1              9.45               0
                                                                                                                       sterically imposed by amino acid sequence alone, as it, appears
1-Carboxymethylhistidine                                                0.69                                0
e-Dicarboxymethyllysine                                                 0.50                                0
                                                                                                                       similarly unreactive in urea-treated metMb (8).
                                                                                                                          It is true that the apparent coincidence of t’he increased num-
      * These analyses   were                       performed      by Professor          F. R. N.          Gurd        ber of imidazoles reacting with Hf ion and bromoacetate upon
and     Dr. L. Banaszak.                                                                                               heme removal from metMb is not absolute proof of the identity
                                                                                                                       of the residues exposed to both reagents.                   Although     the lack
of the relative        conformations         of native    globin and carboxy-                                          of reactivity of the Fs of metMb to both Hf ion and bromo-
methylated native globin by other criteria has been initiated by                                                       acetate, and its release in globin, is reasonable, the lack of FG3
ultracentrifugal       studies. A 1 7O solution of carboxymethyated                                                    reactivity     is an enigma.       Some additional           evidence obtained
native globin at pH 7.5 in 0.16 M KC1 migrated at 25” as a single                                                      from hemoglobin mutants, however, would seem to strengthen
symmetrical peak with szo,W = 1.73 compared with an average                                                            the case for the identity of the residues reacting with both rea-
value of szo.w for globin under similar conditions of 1.78.g From                                                      gent,s. The El residue of metMb is linked in its N-3 position
the molecular weights of the two proteins, the frictional ratio,                                                       to the Fes+-bound HzO, and otherwise appears buried inside the
f carborgmethylated globin lfglohin,     may be calculated as 1.09. These                                              protein.     Diminished      reactivity to bromoacetic acid and con-
results suggest some conformational              change or reaction of globin                                          ceivably a low enough log k’ to place it in the category of im-
with bromoacetate,           but one apparently          insufficient to expose                                        idazoles masked to Hf ion might therefore be ant.icipated.                   Hom-
methionyl residues or to cause the polymerization                  noted for acid-                                     ever, it appears to react in metMb at least after long exposure t,o
denatured globin (22). Moreover, the similarity in sedimenta-                                                          bromoacetic acid (8). The conformational                  similarity of metMb
tion of metMb and carboxymethyl-metMb                       has also been noted                                        with the individual chains of methemoglobin                is known (3, 231, as
 (8) and consideration           of the difference in molecular weight of                                              is t’he presence in metHb of a large number of imidazoles buried
these two proteins suggests t,hat a similar minor conformational                                                       to H+ ion (20). In hemoglobin,                therefore, the histidine cor-
change may occur on reaction of metMb with bromoacetate.                                                               responding t,o the ET of metMb would also be expected to be
      It would appear, then, that removal of heme from metMb                                                           unreactive to H+ ion. A hemoglobin                    mutant, Hb Zurich, in
results in the exposure of 3 additional histidines to bromoacetic                                                      which this hi&dine        is replaced by arginine, is spectrally similar
acid and presumably to solvent.                 The approximate        correlation                                     to normal Hb and presumably                involves      no ma,jor     change    in
of this conclusion with that obtained from titration                       data is                                     heme-protein       attachment.       That CN--metHb              Zurich is elec-
gratifying and also adds strength to the thesis that the additional                                                    trophoretically     inseparable from normal CN--metHb                  at pH 6.5
histidines released to bromoacetate are those exposed to H+ ion                                                        while migrating more slowly towards the anode at pH 8.5 (24)
in the unalkylated protein.              The identity of these histidines may                                          would seem only to be explicable in terms of an essentially normal
therefore be tentatively deduced from comparison of the alkyl-                                                         log k’ for that histidine in normal hemoglobin.                    To the extent
ated residues in globin and metnIb.                  In metMb, the 4 histidyl                                          that metHb and metMb structures are similar, then, the unex-
residues that have been determined                    as unreactive to bromo-                                          pected reactivity of ET to bromoacetate in metMb may be corre-
acetate are the C1, Fs, FG3, and EFI or EFs, whereas only FGI                                                          lated with its reactivit,y to H+ ion in metHb.              Moreover, this cor-
is unreactive in globin.             The release of Fg upon heme removal                                               relation suggests that on removal of heme from globin, only 1 or
would be anticipated,              as nitrogen atom 3 of this histidine is
                                                                                                                          lo These conclusions   are based      upon study of a large scale model
   g The calculated                        value     of ~20,Wfor   globin appears to be slightly                       of metMb,    built from the Kendrew         structure,    in the laboratory     of
lower in KC1 alone                         than    in potassium     phosphate         buffer.                          Professor   F. R. N. Gurd.
February      1964                                                         IS. Breslow                                                                   493

 remotely possibly 2 additional histidines, t,he Fs and possibly the
 FG3, should be released into H+ ion equilibrium                in the absence
of conformational        changes. From the titration data alone, then,
 the increased reactivity of 3 histidines to H+ ion is indicative of
a conformational        difference between globin and metMb.
    It is interesting to note here that the shapes of the titration
curves of globin and metMb also suggest another means by
 which their relative helix contents might be assessed. The                         *
greater stability of myoglobin derivatives than of globin to acid                    b      25                GLOBIN(expt’l)
                                                                                    -                ---GLOBIN(theoretico
 (and also to alkali) is undoubtedly            due in part to stabilization
of the native structure by heme. It appears in Fig. 3, moreover,                        X

that metMb denaturation              occurs over an appreciably narrower                 :: 20
pH range than does that of globin.               If the denaturation    may be
viewed rather simply as a polypeptide helix + coil transition,                      7
then the treatment of such transitions, as for example by Schell-                           15
man (25), may be cited to indicate that the broadening                     of the
transition in globin could be due to a decreased number of resi-
dues involved in the unfolding process as well as to the absence of
heme stabilization.           It would seem, therefore, that a detailed
study of the denaturation           of both globin and met&lb could lead

                                                                                                                                                                 Downloaded from by guest, on March 25, 2013
to an estimate of their relative helical contents.
    Phenolic Equilibria-In          a preliminary     communication     (7), the
ionization of the 3 globin tyrosines was compared with the data of                           0
Hermans (15) for phenolic equilibria               in CO-Mb.      The marked                         B              9          IO        II   12   13
difference observed between the two proteins was most simply
attributable    to the exposure in globin of the tyrosyl residue found
buried in CO-Mb, although it was noted that a possible conforma-                        FIG. 8. Spectrophometric     titrations of tyrosines in globin, CO-
                                                                                     Mb, and CN--metMb         in glycine-KC1 buffer, ionic strength O.lG.
tional change occurred in globin within the pH range of tyrosine                    At 25”: n , globin; 0, CO-Mb; 0, CN--metMb;             - - -, theoretical
titration.     The relative tyrosine titration curves of globin and                  globin titration   (see the text).     At 9”: A, CN--metMb.       Arrows
several myoglobin derivatives have therefore been re-examined                       indicate pH regions in which increases in optical density could be
under identical conditions.              In Fig. 8, the change in molar              observed during a 30-minute interval.
extinction at 245 rnp in glycine-KC1 buffer, ionic strength 0.16,
is shown for globin, CO-Mb, and CN--metXb                    at 25” as well as       mately the effect of two negative charges in the Mb derivatives
for CN-metMb           at 9”. The not unexpected similarity               of the     not present in globin, assuming the similarity of log k’ for lysine
CO-Mb and CN--metMb                tit,rations is readily apparent, and the          in the different proteins.“)    It is easily seen that below pH 11 a
discrepancy betlveen globin and Mb phenolic equilibria is similar,                   large discrepancy exists between the observed and theoretical
although not quite as striking, as that previously deduced from                      globin tyrosine ionizations; i.e. globin titration cannot be simply
comparison of globin t’itrations from this laboratory with CO-Mb                     accounted for by the introduction      of another tyrosyl residue into
titrations from Hermans.                                                             H+ ion equilibrium      in the native protein.    If indeed the third
    As previously cited, the degree of globin ionization is independ-                tyrosine is in Hf ion equilibrium     in native globin, a shift in log
ent of t,ime within the entire pH range studied, and the titration                   k’ of the other ionizable tyrosines must also be assumed to occur
curve is essentially reversible from pH 13, although a slight shift                  upon heme removal.
to approximately         0.2 lower pH unit betn-een pH I1 and 12 on                      il considerable amount of evidence can be accumulated to she\\
back-titration      is discernible (7). In the 1lb derivatives, hom-                that the upper third of the globin tyrosine titration           curve is
ever, time-dependent         increases in optical density can be observed            accompanied by a conformational         change.    The ~~0.~ of a 1%
above pH 12, becoming more rapid as the pH is raised. Above                         globin solution in 0.16 M KC1 increases sharply from approxi-
this pH, the extent of optical density increase at 245 rnk can be                    mately 1.8 at pH 11 to 2.1 at pH 11.X at 25” with no subsequent
generally correlated with the extent of denaturation               (specifically    increase at more alkaline pH.          Moreover, increasingly     dimin-
observed here by optical density changes at 350 mp), an observa-                    ished solubility on return to pH 7 occurs as the pH of a globin
tion compatible with Hermans’ thesis that 1 tyrosyl residue in                      solution is raised above 11, although even after exposure to pH
n-hale CO-Mb is masked to solvent in the native protein.                    -Us0     13, only a fraction of the total globin is precipitated       at pH 7.
shown in Fig. 8 is a theoretical curve for globin tyrosine ionization                More dramatic evidence of a major globin conformational         change
constructed by use of the assumption that all 3 globin tgrosines                    occurring at 25” above pH 11 is seen from a study of the optical
are titratable in the native protein with log k’ equivalent to that                 rotation at the sodium D-line with change of pH. At 25”, a
of the 2 titratable tyrosines in the Mb derivatives.                Specifically    small decrease in [o(h, from -18” at pH 7 to -21” at pH 11
t’he curve was constructed by using the equation                                    occurs, followed by a sharp decrease to -45” at pH 12 and to
                                                                                      -50” at pH 12.5. No further decrease above this pH is demon-
                        AE, lob in = 3/2 A&O-Y,>                             (3)
                   (at pH X - 0.1)       (at pH X)                                     I1 Actually, preliminary  calculations  suggest that log X-’ for
                                                                                    lysine may be slightly higher in metMb than in globin.    Consider-
where AE denotes increase in optical density.    (The shifting of the               ation of this would shift the theoretical globin curve to still more
theoretical curve to 0.1 lower pH unit is to accommodate approxi-                   alkaline pH.
                                             Changes Accompanying             Heme Binding        to Apomyoglobin                                  Vol. 239, No. 2

                                                                                                                -AH(2’2     -    T,)
                                                                                            pHz   -   PHI   =                          + 0.868 (WI.% -    w,.%)       (5)
                                                                                                                   2.3R   TaTI

                                                                                         The value of AH for tyrosine ionization, so calculated, should
                                                                                    be insensitive to differences in charge and shape between the two
                                                                                    temperatures.         Q’, however, would reflect such differences and,
                                                                                    in the pH region of normal tyrosine ionization, should gradually
                                                                                    decrease with pH because of the higher enthalpy of lysine ioniza-
                                                                                    tion and the consequent larger negative charge on the protein
                                                                                    with increasing temperature                 at the same degree of tyrosine
                                                                                    ionization.      It can immediately be seen from Fig. 9, however,
                                                                                    that near 60% ionization a significant increase of titration curve
                                                                                    pH displacement occurs, indicating                 an actual increase in Q’ at
                                                                                    higher pH. Between 10 and 50% ionization,                      an average value
                                                                                    of Q’ between 25” and 9” or between 41” and 25” may be cal-
                                                                                    culated from Fig. 9 as 5.0 kcal or 4.6 kcal, respectively.                  More-
                                                                                    over, as shown in Fig. 10, no significant change in globin con-
                                                                                    formation at 25” and 9” is demonstrable                     by optical rotation
                                                                                    below 60% tyrosine ionization.                 Assuming a value of u = 0.068
                                                                                    at these two temperatures within this pH range, and calculating

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                                                                                     2 at the two temperatures from titration data (see (7) and Fig.
                                                                                     3), AH below 60% tyrosine ionization                 can be calculated as 6.6
   FIG. 9. Tyrosine ionization in glycine-KC1 buffer, ionic strength                 kcal, in keeping with the usually assigned value of 6 to 7 kcal
0.16 at 41” (O), 25” (0), and 9”(A).       All values of percentage                  for t,he AH of tyrosine        ionization.        At 41”, approximately        half
ionization were calculated at each temperature from the changes                      the globin is already denatured below pH 10.8 (see the legend for
in molar extinctions at 245 rnp, assuming no tyrosine ionization                     Fig. 10). By using a value of w = 0.055 (intermediate                    between
below pH 7 and 100% ionization      at the pH values shown, above
which no significant optical density increases seemed to occur.                       the values for the native and acid-denatured              species), an average
The average value of AEw was 11,500 per tyrosine.                                   AH may be calculated from the titration                  data (see (7) and Fig.
                                                                                      3) between 10 and 50% ionization as 7.1 kcal, suggesting that
                                                                                      the tyrosines ionizing in this pH range are almost equally avail-
strable.    The change in rotation appears instantaneously            upon
                                                                                      able in native and denatured conformations.                  Near 83% ioniza-
increasing the pH, and is at least partially and instantaneously
                                                                                      tion, however, the value of Q’ has become 9.8 and 11.3 kcal
reversible upon lowering the pH.           In two separate experiments
                                                                                      between 25-9” and 41-25”, respectively.                  Moreover, it can be
at 25”, the rotation of a globin solution in 0.16 M KCl, exposed
                                                                                      shown (Fig. 10) that at both 25” and 9”, the increase in optical
 for 16 hours to pH 12 and 13, fell immediately to -27” and -37”,
                                                                                     levorotation     with pH occurs in a pH range coincident approxi-
 respectively, upon return to pH 10.8. In one experiment                   in
                                                                                      mately mith the last third of the total tyrosine titration,                   and
 which globin was exposed to pH 12.5 for only 20 minutes, the
                                                                                      therefore with a midpoint near 83% ionization.                   For the same
 rotation on return to pH 10.8 was also -27”.                  No further
                                                                                     degree of ionization        u-ithin this temperature           range, then, no
 decrease on prolonged exposure to pH 10.8 was observed.
                                                                                     large difference in w at the two temperatures should be expected,
    The approximately     parallel correlation of the optical rotation
                                                                                     and the increase in Q’ would seem to be a reflection of a real
 change with the last third of the globin tyrosine titration curve
                                                                                     increase in AH of tyrosine ionization               (see Equations 4 and 5).
 at 25” is a suggestive but not conclusive indication that 1 globin
                                                                                      Presumably the discrepancy in Q’ near 83% ionization,                       when
 tyrosine may also be masked in the native protein and only
                                                                                      calculated within the higher and lower temperature ranges, is a
 become ionizable upon denaturation.           The possibility must still
                                                                                     reflection of the fact that at 41”, at the lowest pH at which it was
 be considered that the coincidence of the conformational           change
                                                                                      possible to obtain optical rotations (Fig. lo), the observed rota-
 with tyrosine ionization     is fortuitous and that all 3 tyrosines
                                                                                      tion indicated considerable denaturation              when only 50% of the
 are merely equally available in native and denatured                st,ates.
                                                                                      tyrosines were ionized.         A difference in w for equivalent degrees
 Evidence to the contrary, however, may be gleaned from the
                                                                                      of tyrosine ionization may therefore contribute to the increased
 comparative globin tyrosine t’itration curves at 9”, 25”, and 41”
                                                                                      value of Q’. In the main, however, the simplest conclusion
 (Fig. 9). The pH displacement between temperatures                 T1 and
                                                                                      invited by the above data is that part of the total tyrosine titra-
 Tz of a given degree of tyrosine ionization      is related to the appar-
                                                                                      tion is conformationally         dependent; or, more specifically, that
 ent heat of ionization,r2 Q’, by the equation (13)                                   1 of the 3 globin tyrosines is ionizable only in the denatured
                                         -&‘Wz     - T,)                              protein.
                       pHz - pH1 =                                                       The apparent reversibility             of globin tyrosine titration       upon
                                            2.3R   T2T,
                                                                                      return from pH 13 (7) remains to be considered.                  Admittedly,      if
and to the intrinsic      heat of ionization,      AH12, by the equation              1 globin tyrosine is unmasked upon globin denaturation,                          an
                                                                                      appreciable shift in the observed tyrosine titration to more acid
    I2 As defined here, the apparent        heat of ionization, Q’, is related        pH should be apparent upon back-titration                 from pH 13. That
to the intrinsic      heat of ionization,   AH, as the apparent    association
constant      for H+ ion at a given DH (loa k’ - 0.868 wz) is related          to     this does not occur is most probably attributable               to reversibility
the intrinsic     association   cons&t     (iogk’).    Equatiod 5 may there-          of denaturation      under the titration conditions.            As has already
fore be derived        directly  from Equations     4 and 2.                          been cited, optical rotation criteria suggest that at least two-
February 1964                                                                           E. Breslow

thirds of the alkaline denaturation       is instantaneously    reversible                           100
upon lowering the pH.         Quite possibly, under titration condi-
tions, in which the protein concentration         is more dilute than in
optical rotation studies, reversibility     is essentially complete.      It
is also possible that failure to observe complete restoration of the
initial optical rotation upon back-titration         is due to imperfect
refolding of the molecule in areas not adjacent to the abnormal
tyrosyl residue.       The assumption will therefore tentatively         be
made that at 25” and go, the upper third of the globin tyrosine
titration    curve is accompanied by a rapid and reversible con-
formational change which releases a previously masked tyrosine
into H+ ion equilibrium.
     If the abnormal globin tyrosine is identical with that found
buried in myoglobin derivatives, the question remains as to the
underlying     cause of the striking differences in globin and myo-
globin tyrosine ionizations.       To a certain extent, of course, this
difference is a reflection of the diminished stability of globin to
alkaline pH. But it is apparent from Fig. 8 that if only 2 tyro-
sines are in Hf ion equilibrium       in native globin (below pH 11)
and in native myoglobin (below pH IL’), a significant decrease in

                                                                                                                                                                                                       Downloaded from by guest, on March 25, 2013
log k’ of these 2 tyrosines occurs when the heme is removed.
Hermans (15) has concluded that in CO-Mb 1 of these 2 tyrosines
has a higher log k’ than the other, but he did not take into account
 the effect of protein charge on pK, in calculating these data.
Assuming, therefore, the identity         of log k’ of both ionizable
 tyrosines and a value of AE245 per tyrosine of 11,500 for both                                             7/        0         9          IO         II          12         13        14 -
proteins, the apparent ionization        midpoint of the tyrosines at                                                                           PH
25” can be estimated as 10.2 in globin and as 11.1 in myoglobin                                      FIG. 10. Correlation           of optical        rotation       changes      in globin       at
derivatives.      Moreover, the potentiometric        titration curves of                       alkaline     pH with tyrosine             titration       curves.       The ionic      strength
CN--metMb         and CO-Mb can probably be assumed to be identi-                               for all studies        was 0.16.     The upper 40% of the spectrophotome&ic
cal with those for metMb at 25” between pH 10 and 12 (13), and                                  tyrosine      ionization      curves     are shown for 9”, 25”, and 41” (--).
                                                                                                At 9” (0)         and 25” (o),        the optical         rotations      are plotted       as the
2 for these proteins can therefore be calculated within this pH                                 percentage        of the total rotational            change at the sodium            D-line       in
range. With a value of w = 0.06 for both globin and myoglobin                                   alkali,    assuming       no change near pH 7. At 41” (A), rotations                            are
derivat.ives, Equation 2 can be used to calrulate log k’ for these                              plotted     as percentage        of the total change from pH 10.85, the lowest
 2 tyrosines as approximately     9.9 in globin and 10.5 in myoglobin                           pH at which           it was possible         to obtain       readings,     although       [(Y], at
                                                                                                 this pH at 41” was -35”              as compared            with -15 to -18”         observed
derivatives.      This latter value is in good agreement with that
                                                                                                for globin samples          near pH 7 at 9” and 25”.
found by Hermans for the 2 reactive tyrosines of guanidinated
 metMb.i3      Although both values of log k’ fall within the range
often described as “normal”       for protein tyrosines, the difference                         are directly linked to the heme (3). Conceivably,           then, this
between them is significant.                                                                    difference is due to an increase in dielectric in the vicinity of the
    Further evidence supporting a difference in log k’ between the                              ionizable tyrosines when the heme is removed.          Whether hhis
2 freely titratable tyrosines of globin and CN--metMb              may be                       alteration   in dielectric is due to conformational      changes ac-
found in the relative values of Q’ for the two proteins between                                 companying      heme withdrawal    or whether 1 of the ionizable
25’ and 9”. From the data in Fig. 8, Q’ for CN--metMb               can be                      tyrosines is close enough to the heme to be directly influenced by
shown to rise gradually from 5.2 kcal at values of AE2d5 x 1OP                                  an expected local dielectric change when the heme is removed
near       6, to 6.4 kcal   at values    near      18 (a pH    region    in which     CN--      cannot be stated at the present time.
metMb is completely               stable and in which the third tyrosine may                        For emphasis, several pertinent comments should be added,
be presumed buried).                By using a value of 11,500 for AEea5  per                   some of which are of particular relevance to the interpretation
tyrosine       in globin,   the   data   in Fig.     9 may    be recalculated        in the     of tyrosine titration curves. It would seem from the data that
same AEt45 interval to show a decrease from 5.2 to 4.2 kcal.                                    neither seeming reversibility,   lack of time-dependent       changes,
Although       the differences in pH displacement     represented by                            nor marked titration curve inflections are a necessary indication
bhese differences in Q’ are only of the order of 0.05 pH unit, the                              of the accessibility         of all tyrosines        to solvent        in the native    protein.
reproducibility    of the results, coupled with the expected decrease                           Rapid and reversible equilibria between two conformations must
in tyrosine    Q’ (in globin)    as the pH is increased,   lends additional                     be considered.    Second, if the difference in tyrosine titration
 support    to a difference   in log k’ between   at least 1 of the 2 titrat-                   between globin and myoglobin is indeed due to a decrease in
able tyrosines        in the two proteins.14     In metMb,      no tyrosines                    log k’ of the freely titratable tyrosines, it is interesting to note
                                                                                                how a difference of 0.6 in log k’ can be appreciably magnified by
    u J. Hermans,         Jr., personal        communication.
    14 Spectrophotometric           titrations       of metMb      itself were also con-        of Fe(OH)2+     ionization        (log k’ = 8.9 at 25”), further     investigation
ducted     at 245 rnp and indicated             a large shift in the apparent          titra-   is required   to ascertain         whether     any of the apparent      shift is due
tion curve       to more acid pH relative               to CN--metMb.            Although       to an unexpected          difference      in tyrosine   acidity  between       metMb
some of this difference          may be attributed            to the ultraviolet      effects   and CN--metMb.
496                                       Changes Accompanying          Heme Binding            to Apomyoglobin                                 Vol. 239, Ko. 2

 the effect of protein charge to greater differences in apparent                 Acknou&dgments-The          author   is particularly    grateful to
acidity.    Conceivably,, large differences in apparent react.ivity           Professor Frank R. N. Gurd for his gifts of sperm whale myo-
of tyrosines in relat.ed proteins to reagents other than Hf ion               globin, and to both Professor Gurd and Dr. L. Banaszak for the
 may also arise from smaller differences in intrinsic reactivities.           amino acid analyses and peptide mapping of carboxymethylated
Finally, it should be emphasized t,hat although 1 tyrosine has                globin.     The able technical assistance of Miss Priscilla Anderson
been concluded to remain masked when the heme is removed                      is also gratefully acknowledged.
from myoglobin, the reversibility      of the globin tyrosine titration
curve cannot make this conclusion unequivocal without demon-                                                     REFERENCES
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myoglobin and its derivative, globin, suggest that at least 3                           (1963).
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