Carp Hemoglobin

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					THEJOURNAL BIOLOGICAL
         OF         CHEMISTRY
Vol. 255, No. 20, Issue of October 25. pp. 9800-9806,1980
Printed in U.S.A.


Carp Hemoglobin
11. THE ALKALINE BOHR EFFECT*

                                                                  (Received for publication, November 21, 1979,and in revised form, May 14, 1980)

                 James C. W. Chien$ and Kevin H. Mayo@
                 From the Departmentof Chemistry. Materials Research Laboratories, TheUniversity of Massachusetts, Amherst,
                                                            _ ,

                 Massachusetts 01003 .



   The Bohr effect of carp hemoglobin has been deter- effects of horse and mouse hemoglobins with HbA’shows that
mined by differential titration, by direct acid-base ti- they are essentially the same (7).
tration, and by calculation from precise oxygen equilib-        Previous studies have shown that the alkaline Bohr effect
rium data over a wide pH variation. The results for the in some of the nonmammalian hemoglobins (8-12) is different
hemolysate and the two major components are essen- from that of the mammalian hemoglobins. In thecase of trout
tially identical. At pH 6 9 in the vicinity of maximum hemoglobin, component I shows little or no Bohr effect
                             .
cooperativity and maximum Bohr effect, the protein          whereas component IV releases up to 8 Bohr protons (13).
releases 3 7 protons in theabsence of added ions. This Detailed study of the Bohr effect for carp hemoglobin has not
             .
Bohr curve above pH 7 is not changed by the presence been reported. The central purpose of
                                                                                                            this work is to present
of 0.05 M 2,2-bis(hydroxymethy1)-2,2’,2’’-nitrilotri-
ethanol (bis-’his) buffer, but is changed below the pK      the results of a thorough investigation of the carp hemoglo-
of the bis-Tris amine, giving a maximum of 4.3 protons bin’s Bohr effect. The results should be valuable to the un-




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at pH 6.65. In 0 1 M phosphate, the maximum is in- derstanding of oxygen-linked properties in teleost fish hemo-
                   .
creased to 6.1 protons and shifted to pH 7 2 . Addition globins and of the phenomenon of the Bohr effect in general.
                                is          .5
             Ps-inositol lowers the magnitude of the Bohr
of 1.4 l l l ~
effect and shifts its maximum to aneven higher pH. At                           EXPERIMENTAL      PROCEDURES
the limit of high pH ( . 2 , without buffer or in bis-Tris,
                        90)                                    Materials-Carp hemoglobin was purified anditscomponents
there is a net uptake about 0.5 proton upon oxygen- separated by procedures described in first paper (14). Even though
                          of                                                                        the
ation. The average heat ionization of the Bohr groups the proteinisextremelysusceptible
                             of                                                                         to autoxidation under most
is 5500 2 800 cal.Even though chloride ion hasa             experimental conditions (15), the protein is stable in deionized water
pronounced effecton the oxygenation properties of foroveraday.Differential                  titrations and direct titrations do    not
                                                            require the reductase system (14)
carp hemoglobin, it has a small influence on the Bohr ments of oxygen equilibrium. The as was necessary in the measure-
                                                                                                   methemoglobin concentration re-
effect up to 0.5 M NaCl. In 5 M NaCl, the magnitude of mained a t 2 to 3% during thetitrations as it was in the initial
the Bohr effect is reduced by approximately 30%. Acid preparation.
base titrations give three to four  oxygen-linked groups       Direct Titration-All samples were approximately 0.6 n m in heme;
for carp hemoglobin in water; thisis increased to about the titrationwas done on 5.0 ml of unbuffered and Con-free solutions
six groups in 2 5 M NaCl. The results suggest that carp of carp hemoglobin. Standardization of 0.1 M NaOH or 0.1 M HCl to
                 .
hemoglobin is functionally versatile and may provide pH 7.0 in Con-free deionized Hz0 and           dilution withCOn-free deionized
one way to regulate its COz transport via heterotropic H,O to 0.01 M were both done prior to each titration. Temperature
                                                                             to within kO.05’C; pH measurements
allosteric interactions. In phosphate buffer at the pH was controlled 476050 combinationelectrode usingwere made with
                                                            a Coming No.                                                 a Radiometer
value where carp hemoglobin is strongly cooperative, research pH meter, PHM6 4 , with the pH read to the nearest                +0.002
the proton releaseis linear with respect to ligand sat- pH unit. Titrations      were done on both oxy and deoxy samples by first
uration. Lowering the cooperativity by either an in- adjusting the pH to 9.2 and titrating in microliter quantities with
crease or decrease in pH results in nonlinear relation- standardized HCl to pH 5.4, or by first adjusting the pH to 5.4 and
ships.                                                      titrating with standardized NaOH to pH9.2.All measurements were
                                                                             made on hemoglobin samples in deionized Hz0 where carp hemoglo-
                                                                             bin was found to be the most stable to denaturation or autoxidation.
                                                                             The pH change versus microliters of acid or base was recorded. IHP,
  Human hemoglobin releases about 2.4 protons per tetramer                   when used, was added only in 10%excess to minimize any buffering
upon oxygenation at physiological pH (1, 2). Kilmartin and                   action from it.
Rossi-Bernardi (3) showed that the a-amino group of the a-                     Differential Titration-Lill samples were approximately 0.6 m in
                                                                                                                                             M
                                                                             heme; the titration was done on 5.0 ml of unbuffered, Cop-free carp
chain contributes 25 to 30%of the alkaline Bohr effect (4).                  hemoglobin. Each solution was deoxygenated with nitrogen   saturated
The combined effects of chemical modification (2, 5 ) and x-                 with water; deoxygenation was continued until no further pH change
ray determination (6) suggest that His-146P could provide                    was detected. The carp hemoglobin was then reoxygenated until the
half of the alkaline Bohr effect. Comparison of the Bohr                     pH stabilized. Microliter amounts of standardized NaOH (approxi-
                                                                             mately 0.01 M) or standardizedHCl(approximately        0.01 M ) were
   * The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby              ’ The abbreviations used are: HbA, normal adult human hemoglo-
marked “advertisement” in accordance with 18 U.S.C. Section 1734             bin; IHP, P6.inositol; bis-Tris, 2,2-bis(hydroxymethyl)-2,2’,2”-nitrilo-
solely to indicate this fact.                                                triethanol; Hbc, carp hemoglobin; k,,Adair constant for i-th stage of
   $ To whom all inquiries should be addressed.                              oxygenation; nmax,     maximal slope of the Hill plot; P,, medium ligand
   8 In partial fulfillment of the requirement for the Ph.D. degree a t      activity; A X , , total number of Bohr protons; AX,, number of Bohr
the University of Massachusetts. Present address, Max Planck Insti-          protons released upon oxygenation of i-th heme; Y , fractional satu-
tut fur Biochemie, Martinsreid bei Munchen, West Germany.                    ration.

                                                                         9800
                                                                             1
                                                            Carp Hemoglobin. 1                                                      9801
addeduntil the pHwas that of the deoxy sample. Prior to each                 of A X l . Thevalues calculated by Equations 1 and 4 are
differential titration, the initial pH was adjusted with either 0.1 M acid   compared in Figs. 2 to 4 and arefound to be ingood agreement
or base.                                                                     except for some deviation at very low pH values.
                                  RESULTS                                                            DISCUSSION
   Differential Titration-The number of protons released at               There are three equivalent methods for the measurement
a given pH upon conversion of deoxyhemoglobin to oxyhe- of the Bohr effect. The simplest and most direct method is
moglobin was determined by differential titration in the ab- that of differential titration. It can be performed in deionized
sence of added ions. The maximum number of protons was water, thus avoiding all possible complications deriving from
observed at approximately pH (Fig. 12).There is an uptakeeither specific ioneffects or the use of a different buffer
                                    6.9
of protons above pH upon oxygenation of carp hemoglobin. system. It measures directly the changes in bound protons
                         8.6
The log Po.5uersus pH resultsof Noble et al. (16,17) have too with either oxygenation or deoxygenation. There is no need
much scatter in this                                a
                         region to observe such phenomenon.             to convert the data into another form such in the case of
                                                                                                                         as
   NaClwas added to the solution            of carp hemoglobinin        direct titration or to assume the validity of linked function
deionized water. Thereis an insignificant NaCl effecton Bohr equations and the invariance the shapeof the oxygenation
                                                                                                        of
proton release for NaCl concentrations 50.5 M; in 5 M NaCl curve with pH order to compute Bohr
                                                                                        in                          effect from the latter
there is a 33% reduction. The uptake of proton in alkaline data. In the               case of carp hemoglobin, differential titration has
solution is seen at all salt concentrations.                            the further advantagesof requiring very little time for meas-
   The Bohr effect was determined at 10" and 20°C. The urement which is important for this unstable protein.
                                            in
results for the hemolysate are shown Fig. 2; the results for               The second and most generally employed method for the
components I and I1 are virtually superimposable to those of measurement of the Bohreffect is to calculate it                  from the pH
the hemolysate (Fig. 1).                                                dependence of oxygenation equilibrium through the linkage
   Direct Titration-The acid-base titration curves of hemo- equation 1. Even though the method has those disadvantages
globins have special significance in relation to the Bohr       effect. mentioned above, it does afford, simultaneously, information




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The titration curves for carp oxy- and deoxyhemoglobin at about the heme-heme interaction andligand affinity. Also,    the
20°C in deionized water are shown in Fig. 3. The profound when coupled with an Adair analysis, one can estimate the
                                                 of
effect of NaCl on the acid-base properties carp hemoglobin stepwise release of Bohr protons.
is given in Fig. 4. IHP greatly increases the number protons
                                                           of              Finally, direct titration shares some of the advantages of
bound by carp oxyhemoglobin as can be seen in Figure 5. The differential titration. It also reveals the total acid-base prop-
                             be
direct titration data can converted to the Bohr          effect curve erties of the protein molecule.
                                             as in
(18).The resulting curves are the same Fig. 1for titration                 Because a great deal is known about the alkaline Bohr
curves in Figs. 3 and 4, and as seen in Figure 6c for the case effect for mammalian hemoglobins, and a detailed stereo-
with IHP.                                                               chemical mechanism has      been proposed (21),comparisons will
   Bohr Effect from Oxygen Equilibria-Precise oxygen equi- be made between mammalian and carp hemoglobins in this
librium measurements on carp hemoglobin have been made discussion. The analogy is justified by the fact that the carp
from pH6 to 9 in bis-Tris buffer and in phosphate buffer with hemoglobin sequences (22, 23) when compared with those of
and without IHP(14). From these data, the number Bohr HbAshow a high degree conservation of residues at the a&
                                                              of                                   of
protons can be calculated with the expression                           tetrameric contact, of amino acids in van der Waals contact
                                                                        with the prosthetic group, and of prolines marking thebegin-
                                                                        ning and end of helical segments.
A computer program wasused to calculate A X , (19). The                    Bohr Effect in theAbsence ofddded Ions-The method of
results for all three media are shown in Fig. 6. The error in differential titration in deionized water is essentially uncom-
A X , is --+2.5%.                                                       plicated by ionic effects. In the acidic region, titration may
   Fractional Bohr Protons-The precise oxygen equilibrium introduce a few miUimoles/ml of Na' ion. Cations at such
curves were nonlinearleastsquaresfitted               (14), using the concentrations are not believed to have any effect on the
computer program of Snyder and Chien(19) according to the oxygenation properties of hemoglobins (24). In the alkaline
stepwise oxygenationtheory of Adair (20).The median            oxygen region, a few millimoles/ml ofC1- ion would be introduced
pressure, P,, of Equation 1 is related to theAdair constants, which also shouldnothaveany                      significant anion effects.
kt's (i = 1, 2, 3, 4), by                                               Under theseconditions, carp hemoglobin releases a maximum
                                                                        number of protons per tetramer, al, 3.7 at pH 6.75 (Fig.
                                                                                                                  of
                                                                                                              at
                                                                        1). Carp hemoglobin, therefore, has least four oxygen-linked
                                                                        acid groups. This is compared to the well established GI=
The stepwise liberation of Bohr protons at the i-th step of 2.5 for mammalian hemoglobins.
oxygenation is                                                             The identification of the groups involved in the alkaline
                                                                        Bohreffect in mammalian hemoglobins has beenaccom-
                          A X t= dlog k,/dpH                        (3) plished by a combination of chemical modifications and crys-
The results are given in Figs. 7 to 9. Since by definition              tallographic studies of the alkaline Bohrgroups. By selective
                                     4
                                                                        carbamylation of the NHP-terminalvaline of the a and fl
                             a,=        m,                          (4) chains, Kilmartin and Rossi-Bernardi (3,25)       showed that the
                                    ,=I
                                                                        a-amino groupof the a-chain, but not Val 1/3 amino group,
                                                                                                                      the
we are provided with an additional method for calculation contributes about 25% of the alkaline Bohr effect. However,
                                                           the
                                                                        the NH2-terminal residue of the carp hemoglobin a-chain is
     Figs. 1 to 10 are presented in miniprint at the end of this paper. acetylserine (22), thus eliminating one source the alkaline
                                                                                                                            of
Miniprint i easily read with the aid of a standard magnifying glass.
             s
Full-size photocopies are available from the Journalof Biological       Bohr group for carp.
Chemistry, 9650 Rockville Pike, Bethesda, Md. 20014. Request Doc-          The other group responsible for half of the alkaline Bohr
ument No. 79 M 2351, cite author(s), and include a check or money effect of human hemoglobin is His 146fl. By studying the x-
order for $1.65 per set of photocopies.                                 ray crystallographic structure of NES-hemoglobin (reaction
9802                                                Carp Hemoglobin. 11
of Cys 93p with N-ethylmaleimide),Perutz etal. (6) proposed NaCl, eliminating the alkaline Bohr effect entirely when the
that there was a salt bridge between His 146p in the H helix NaCl concentration reaches 5 M.
and Asp 94p in the F helix, and that thebreaking of this salt         In the case of carp hemoglobin, both proton and chloride
bridge could provide half the alkaline Bohr effect in mam- ions are strongeffectors of its oxygenation properties (14), yet
malian hemoglobin. In both major components of carp he- Fig. 1 showed that up to 0.5 M NaCl is without signifkant
moglobin, the P-chains have His as the     COOH-terminal resi- influence on the      alkaline Bohr effect of carp hemoglobin. Even
due (the p chain has 147 residues). The 94p residue is Glu. in 5 M NaCl, the magnitude of the Bohr effect is reduced by
Looking at the Cambridge model for deoxyhemoglobulin, the only approximately 30%. This can be understood from the
                                                               in
extension of the carboxyl group due to the extra methylene effect of NaCl onthe acid-base properties of carp hemoglobin.
Glu 94p may enable it to form a salt bridge with His 147p.            The acid-base titration results (Figs. 3 to 5) of carp hemo-
   There are other salt     bridges in mammalian hemoglobin globin are compared with those of human hemoglobin (31)in
which could be potential sources for Bohr protons but their Table I. Between pH 5.2 and 9.2, carp hemoglobin has three
                                   be
actual contributions have yet to determined-these are Lys to four titratable          groups per heme ascompared to about nine
40al-His 146p2, Asp 94al-His 146p2, Asp 126al-Arg 141a2,Lys for human hemoglobin. In deionized water, carp deoxy- and
127al-Arg 141a2, Val lP1-His 146p2, Asp 94aI-Asn 102p2 (26- oxyhemoglobin solutions have pH values of 6.0 and 5.7, re-
28). In carp hemoglobin, the COOH-terminal histidine in the spectively; the corresponding values for human hemoglobin
chain is No. 147 in the sequence; however, it is situated at the derivatives are both pH Again, comparing the two species,
                                                                                             6.7.
HC3 position as is the corresponding histidine in HbA. All the carp oxyhemoglobin binds protons less strongly than deoxy-
other amino   acids in the above listare found in carphemoglo- hemoglobin and over a wider pH range than the          corresponding
bin at theidentical location. Therefore, those saltbridges are differences for human hemoglobins. However, at pH values
also potential sources for Bohr protons in carp. In fact, the greater than 8.6, the carp oxy molecule has a larger proton
largeralkaline Bohr effect observedfor carp than human             affinity than the deoxy molecule. This isreflected in the
hemoglobins suggests that some of these interactions maybe uptake of protons observed in differential titration data and
more strongly oxygen-linked in carp.                               the Bohreffect calculated from the pHdependence of oxygen-




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   Carp hemoglobin is devoid of the usual acid Bohr effect at ation curves in bis-Tris (see above).
low pH. The lowest pH reached by the present investigation            The effect of 2.5 M NaCl has profound consequence on the
is pH 5.5; HbA attains the   maximum for proton uptake at this acid-base properties of carp hemoglobins. The number of
pH. On the other hand, where the carp hemoglobin Bohr titratable groups increases to about six. The effects are suff-
titration curve becomes negative above pH 8.6, there is no ciently different for the oxy- and deoxyhemoglobins of carp;
analogous uptake of protons in alkaline media by mammalian as a result, there is still a significant alkaline Bohr effect for
hemoglobins. It seems that thereis a structural change at pH carp hemoglobin in 2.5 M NaCl. At lower NaCl concentrations,
8.6 which exposes some masked basic residues upon oxygen- 0.5 M and less, the influence on the oxy and deoxy states are
ation. In otherwords, above this pH, carp    oxyhemoglobin has probably more comparable rendering the NaCl influence on
a larger proton affinity than the deoxy species.                   Bohr effects too small to be seen clearly by differential titra-
   Influence of Temperature-The influence of temperature tions (Fig. 1).
on the Bohr effect in human and in horse hemoglobin has              Bohr Effect in bis-Tris Buffer-bis-Tris is said to have no
been investigated by Antonini et al. (18).Rossi-Bernardi and specific ion effect on the oxygenation properties of human
Roughton (29) carried out differential titrations of human hemoglobin (32) because the bis-Tris amine is not ionized at
hemoglobin at 25°C and 37°C. The increase in temperature physiological pH. This isborne out for carp hemoglobin.
causes a reduction of at     by 0.3 proton per tetramer shifting Above pH 6.8, the Bohreffect calculated from pH-dependent
the maximum for the Bohr effect from pH 7.3 to 7.0. By oxygenation curves is identical to thatof differential titration
assuming the presence of two oxygen-linked acid groups, the (Fig. 6). Itis also reassuring that the uptake protons above
                                                                                                                   of
authors estimated thepK values forthese groups and a value pH 8.6 was reproduced by the twodifferent methods of
of5000 cal for theirheats of ionization. Thismethod of measuring the Bohr                                       however, markedly
                                                                                         effect. The results are,
analysis (29) cannot be adapted readily to estimate the pK different below pH 6.8. In bis-Tris, a maximum of 4.3 Bohr
values for the Bohr groups because there is a t least one more protons were liberated at pH 6.65. The bis-Tris amine with a
group in carp hemoglobin than in human. Also, it requires pK value of 6.5 becomes protonated; the protonated amine
correction for the heats of oxygenation of hemoglobin in the may exert a specific ion effect to possibly increase the mag-
region where “Bohr effects” have disappeared; this cannot be nitude of the alkaline Bohr effect.
determined for carp hemoglobin.:’ However, one can make a            According to theAdair theory of stepwise oxygenation (20),
crude estimate utilizing the formula derived by Wyman (30)         the number of Bohr protons liberated at the i-th stage of
                    Q = -2.303 RTZ(apH/aZh                    (5)
                                                                   oxygenation (AX,) can be calculated with Equation 3 and the
                                                                   total number of Bohr protons given by Equation 4.Fig. 7
where Q is the apparent heat ionization of the Bohrgroups shows that A X l calculatedfrom Equations 1 and 4 are in
                                of
and (dpH/dnB is the change in pH with temperature of the excellent agreement; the same is true in the other buffers
carp hemoglobin solution containing a fixed amount of base. (Figs. 8 and 9). At low pH inbis-Tris, the majority of the Bohr
From the data in Fig. 2, we find Q = 5500 (+800)cal.               protons are liberated after thebinding of the third and fourth
   Effect of Chloride Zon-The effect of salt on oxygen equi- oxygens, the contributions from A X 1 and A X 2 being much
librium of hemoglobin is a well recognized phenomenon. An- smaller. At the pH of maximum cooperativity, the contribu-
tonini et al. (24) showed that in low salt concentration the       tions of all four stages of oxygenation to the Bohr effect are
effect of decreasing the oxygen affinity appears to be due comparable. At pH > 8.6, uptake of protons occurs mostly
primarily to anions. In this concentrationregion, the recipro- with oxygenation of the thirdheme. The maxima for A X t were
cal effect of salt on human hemoglobin is to displace the found at approximately pH 5.8, 6.4, 6.7, and 7.0 for i = 1, 2, 3
alkaline Bohr effect toward higher pH by 0.8 unit without          and 4, respectively.
decreasing its magnitude. This effect is a continuous one for         Bohr Effect in Phosphate Buffer-Judging from limited
    J. C. W. Chien and K. H. Mayo, manuscript submitted for publi- data on the alkaline Bohr effect for human hemoglobin either
cation.                                                            in deionized water or in bis-Tris buffer and comparing them
                                                          Carp Hemoglobin. 11                                                             9803
                                                                TABLE   I
                                               Acid-base titrations of carp hemoglobins
                                                                            H+         H+               AH+           AH'
                                                                                                H'+'y$p' -
                                                                                                                                  A   H   +



                                       H+        H+                       (Hb,On),   (Hb.Od,"         (Hb,Oz        (Hb.0, -   (HbcO, -
    pH       H+ (Hbc)   +H   (Hbd"   (Hb.Oz)   (Hb.Oz)"    2.5 M(Hbc)p
                                                            +H    NaCl   2,5 NaCl       NaCl            HbJ           Hb*)
                                                                                                                               Hbc)   +       2.5
                                                                                                                                M NaCl

    5.2                  +3.89       +0.72      +4.15                                 +4.57      +2.30                +0.26
    5.4                  +3.33       +0.43      +3.59        +2.29        +2.25       +3.95      +2.09                +0.26      -0.04
    5.6       +0.4       +2.79       +0.08      +3.02        +1.86        +1.67       +3.38      +1.81     -0.32      +0.23      -0.19
    5.8       +0.17      +2.36       -0.24      +2.48        +1.34        +1.06       +2.87      +1.53     -0.41      +0.12      -0.28
    6.0         0        +1.93       -0.57      +1.94        +0.90        +0.47       +2.37      +1.23     -0.57      +0.01      -0.43
    6.2       -0.19      +1.48       -0.85      +1.39        +0.46          0         +1.89      +0.93     -0.66      -0.09      -0.46
    6.4       -0.33      +1.03       -1.08      +0.81          0          -0.47       +1.39      +0.64     -0.75      -0.22      -0.47
    6.6       -0.57      +0.53       -1.40      +0.21        -0.33        -0.90       +0.86      +0.37     -0.83      -0.32      -0.57
    6.8       -0.68      -0.01       -1.61      -0.42        -0.73        -1.32       +0.32        0       -0.93      -0.41      -0.59
    7.0       -0.87      -0.53       -1.74      -1.01        - 1.04       -1.58       -0.25      -0.48     -0.87      -0.48      -0.54
    7.2       -1.08      -1.14       - 1.86     - 1.65       -1.24        -1.76       -0.85      -0.85     -0.78      -0.51      -0.52
    7.4       -1.32      -1.72       -1.98      -2.22        -1.43        -1.90       -1.48      -1.08     -0.66      -0.50      -0.47
    7.6       -1.53      -2.27       -2.07      -2.73        -1.64        -2.04       -2.13      -1.23     -0.54      -0.46      -0.40
    7.8       -1.74      -2.79       -2.17      -3.20        -1.86        -2.18       -2.68      -1.32     -0.43      -0.41      -0.32
    8.0       -1.93      -3.28       -2.24      -3.60        -2.09        -2.30       -3.20      - 1.44    -0.31      -0.32      -0.21
    8.2       -2.14      -3.73       -2.31      -3.95        -2.30        -2.40       -3.62      -1.56     -0.17      -0.22      -0.10
    8.4       -2.27      -4.06       -2.42      -4.22        -2.50        -2.54       -3.98      -1.67     -0.15      -0.16      -0.04
    8.6       -2.49      -4.34       -2.49      -4.45        -2.69        -2.69       -4.28      - 1.79      0        -0.11        0
    8.8       -2.66      -4.63       -2.59      -4.72        -2.91        -2.83       -4.55      - 1.93    +0.07      -0.09      +0.08
    9.0       -2.83      -4.93       -2.73      -4.98        -3.16        -3.11       -4.82      -2.07     +0.10      -0.05      +0.05
    9.2       -3.0       -5.26       -2.87      -5.30        -3.46        -3.40                  -2.25     +0.13      -0.04      -0.06




                                                                                                                                                    Downloaded from www.jbc.org by guest, on May 11, 2010
    Antonini and Brunori (31).
with the abundant results in phosphate buffer, one findsthat             instance, in bis-Tris, these maxima are found between pH 6.4
there is little or no influence by low concentrations of phos-           and 7.0, whereas in phosphate they lie within 0.2 pH unit.The
phate ions. In contrast, phosphate ions have a pronounced                sum of C:=, A X , = 5.4 protons in 0.1 M phosphate; it is 4.7
influence on carphemoglobin by increasing      ut   to 6.1 protons       protons in bis-Tris. The Bohr curve for IHP in Fig. 6 shows
and by shifting the maximum to pH 7.25.                                  that the Bohr protons are distributed over an even wider pH
   Comparison between carp and human         hemoglobins in their        range than in bis-Tris.
stepwise Bohr effect is possible in 0.1 M phosphate since Imai              Proton Release and Ligand Saturation-For many years
and Yonetani (34) have reported the calculation for the latter                                                            of
                                                                         it has generally been accepted that the shape the Hill plot
system. In the case of HbA, the contribution from AX, is only            for mammalian hemoglobin does not change over a wide range
3% of the total proton release at the maximum Bohr effect                of pH variation. This invariance has been given different
and is nearly independent of pH from pH 6.5 to 8.8; therefore,           interpretations (33-35).
all the Bohr protons are liberated when three of the hemes                  Fig. 10 contains plots of u,   uersus Y for carp hemoglobin
have been oxygenated in HbA. The maximum values for AXl,                 in phosphate buffer at several pH values. At pH 6.92, where
A X 2 , and       were observed at pH 7.1, 7.8, and 7.4, respec-         the protein has maximum cooperativity (14), the line is very
tively. These values converge in both very acidic and basic              close to the 45" line for linear relationship. Lowering the pH
media. The most striking difference between carp hemoglobin              results in a concave curve below the 45" line; there are fewer
and human hemoglobin is the strong dependence of A X 4 on                Bohrprotons released for a given saturation. Conversely,
pH for carp. It has a larger value of 2.5 protons at pH 6.2              raising the pH produces convex curves above the linear line
which decreases monotonically to a value of 0.2 proton at pH             indicating more Bohr protons released at anyparticular frac-
9.0. In the region of maximum Bohr effect, the contributions             tional saturation. These results support the conclusions of
are A X z > A X , = A X 3 > A X 4 . The maximum values of A X I ,        Imai and Yonetani (33) and show that a linear relationship
A X 2 , and A X 3 are found at pH 7.4, 7.3, and 7.2, respectively.       between A X , and 7 is not to be expected from hemoglobins
The monotonic decrease of A X 4 with increasing pH implies a             with reduced cooperativity.
continuous change of the structure of carp oxyhemoglobin                    Conclusion-Current interpretation of the alkaline Bohr
with pH.                                                                 effect is based largely on the stereochemical model proposed
   The phenomenon of proton uptake at alkaline conditions                by Perutz and coworkers (6, 21). This entails changes of pH
by carp hemoglobin is eliminated by the presence of the                  values of the various Bohr groups mentioned above upon
phosphate ions.                                                          oxygenation. It is not clear what the relative contributionsare
   Influence of IHP on the Bohr Effect-Addition of IHP to                from quaternary and tertiary structural transitions. For hu-
a 0.1 M phosphate solution of carp hemoglobin causes a                   man hemoglobin, the analysis of oxygenation data by Imai
significant decrease in AX,, shifting their liberation to high pH        (36) showed an allosteric transition to occur after the ligation
values spreading over a very wide pH range, Comparison of                of the third heme. Analysis of the stepwise proton release by
Figs. 8 and 9 showed the following. IHP shifts A X z to a higher         Imai and Yonetani (33) showed that atpH 7.4, A X , = 0.4, A X 2
pH by half a unit and lowers its magnitude and it shifts the             = 0.7, A X 3 = 1.1, and A X 4 = 0. There are about 50% of the
A X 3 maximum from pH 7.2 to 8.3 without changing its mag-               Bohr      liberated
                                                                             protons      throughtertiary                   conformation
nitude. The effect of IHP on AX, is like that of A X 3 except            changes, the remainder being released upon oxygenation of
that it causes a rise in A X 1 below pH 7.5. IHP is almost               the thirdheme accompanied by a quaternary transition.
without any effect on A X 4 , suggesting that IHP is expelled            Therefore, the second 50% of the alkaline Bohr effect may be
from its binding site upon oxygenation of the third heme.                said to be the combined results of tertiary and quaternary
   The difference between AX, found in various buffers is                structural changes. Intermediately oxygenated species of HbA
partly attributable to thepH range for A X , for i = 1 to 3. For         are unstable.
9804                                                       Carp Hemoglobin. I1
   In contrast, A X 4 is large for carphemoglobin except at high           13. Binotti, I., Giovenco, S., Giardina, B., Antonini, E., Brunori, M.,
pH values. As pH decreases, A X 4 increases so that in phos-                     and Wyman, J. (1971) Arch. Biochem. Biophys. 142,274-280
phate buffer at low pH most of the protons are released only               14. Chien, J. C. W., and Mayo, K. H. (1980)J.Bwl. Chem. 265,9790-
                                                                                 9799
after all the hemes are oxygenated. At intermediate pH values,             15. Chien, J. c . W., and Mayo, K. H. (1979) Biochim. Biophys. Acta,
the major contribution to the alkaline Bohr effect comes from                    i press
                                                                                  n
different stages of oxygenation depending upon the environ-                16. Noble, R. W., Parkhurst, L. J., and Gibson, Q. H. (1970) J.Biol.
ment. The f i t versus Y plot (Fig. 10) suggests that interme-                   Chem. 245,6628-6633
diately oxygenated species are stable. The results demonstrate             17. Tan, A. L., and Noble, R. W. (1973) J. Biol. Chem. 248, 2880-
the ability of the carp hemoglobin molecule to modify its                        2888
                                                                           18. Antonini, E., Wyman, J., Brunori, M., Fronticelli, C., Bucci, E.,
structure with changes in its environment.                                       and Rossi-Fanelli, A. (1965) J.Biol. Chem. 240, 1096-1103
                                                                           19. Snyder, R. W., Jr., and Chien, J. C. W. (1978) Eur. J. Biochem.
  Acknowledgements-We are grateful to Drs. L. C. Dickinson and                   91,83-88
F. W. Snyder, Jr. for helpful discussions and to Dr. R. Gunter for         20. Adair, G. S. (1925) J.Biol. Chem. 63,529-538
assistance in computer programming. J . C. W. C. wishes to acknowl-        21. Perutz, M. F. (1970) Nature 228, 726-739
edge the receipt of a Humboldt Award and the hospitality Professor
                                                        of                 22. Hilse, K., and Braunitzer, G. (1968) Hoppe-Seyler’s 2. Physiol.
G . Braunitzer, Max-Planck-Institut fur Biochemie, Martinsreid bei               Chem. 349,433-450
Miinchen.                                                                  23. Grujic-Injac, B., Braunitzer, G., and Stangle, A. (1979) Hoppe-
                                                                                 SeylerS 2. Physiol. Chem. 360, 609-612
                                                                           24. Antonini, E., Amiconi, G., andBrunori, M. (1972) in Oxygen
                            REFERENCES
                                                                                Affinity of Hemoglobin and RedCell Acid-Base Status:     Alfred
 1. Wyman, J . (1948) Adu. Protein Chem. 4, 407-531                              Benzon Symposium IV (Rorth, M., and Astrup, P., eds) pp.
 2. Benesh, R., and Benesch, R. E. (1961) J. Biol. Chem. 236, 405-               121-129, Munksgaard, Copenhagen
      410                                                                  25. Kilmartin, J . V., and Rossi-Bernardi, L.(1969) Nature 222, 1243-
 3. Kilmartin, J . V., and Rossi-Bernardi, L. (1971) Biochem. J. 124,            1246
      31-45                                                                26. Bolton, W., and Perutz, M. F. (1970) Nature 228, 551-552
 4. Kilmartin, J . V. (1972) in Oxygen Affinity of Hemoglobin and




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                                                                           27. Muirhead, H., and Greer, H. (1970) Nature 228,516-519
        Red Cell Acid-Base Status: AlfredBenzon Symposium IV               28. Perutz,M. F., Ladner, J. E., Simon, S. R., and Ho, C. (1974)
        (Rorth, M., andAstruip, P., eds)Munksgaard,pp.           93-100,         Biochemistry 13,2163-2173.
        Copenhagen                                                         29. Rossi-Bernardi, L., and Roughton, F. J. W. (1967) J.Biol. Chem.
5.    Riggs, A. (1961) J. Biol. Chem. 236, 1948-1954                             242,784-792
6.    Perutz, M. F., Muirhead, H.,Mazzarella, L., Crowther, R. A.,         30. Wyman, J. (1939) J. Biol. Chem. 127, 1-13
        Greer, J., and Kilmartin, J. V. (1969) Nature 222, 1240-1243       31. Antonini, E., and Brunori, M.(1971) Hemoglobin and Myoglobin
 7.   Smith, D. B., Brunori, M., Antonini, E., and Wyman, J . (1966)             in Their Reactions with Ligands, pp. 106-107, North Holland
        Arch. Biochem. Biophys. 113, 725-729                                     Publishing Co., Amsterdam
 8.   Manwell, C. (1958) Science 127, 705-706                              32. Benesch, R. E., Benesch, R., and Yu, C. I. (1969) Biochemistry 8,
 9.   Manwell, C. (1960) Annu. Rev. Physiol. 22, 191-196                         2567-2571
10.   Antonini, E., Wyman, J., Bellelli, L., Rumen, N., and Siniscalco,    33. Imai, K., and Yonetani, T. (1975) J. Bwl. Chem. 250, 2227-2231
        M. (1964) Arch. Biochem. Biophys. 105,404-414                      34. Wyman, J . (1939) J.Biol. Chem. 127,581-599
11.   Riggs, A. (1965) Physiol. Rev. 45,619-624                            35. Roughton, F. J. W., Otis, A. B., and Lyster, R. L. J. (1955) Proc.
12.   Brunori, M., Antonini, E., Wyman, J., Tentori,L., Vivaldi, G., and         Roy. SOC. Lond. B Biol. Sci. 144, 29-38
        Carta, S. (1968) Comp. Biochem. Physiol. 24,519-527                36. Imai, K. (1973) Biochemistry 12, 798-808
                            9805




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