THEJOURNAL    OF BIOLOGICAL  CHEMISTRY                                                               Vol. 261, No. 18, Iss...
                                       Properties              of Lumbricus terrestris
8416                             Oxygenation Properties of Lumbricus terrestris Hemoglobin

Oxygenation Properties of Lumbricus terrestris Hemoglobin                                                      8417



                                     Oxygenation Properties of Lumbricus ...
Oxygenation Properties of Lumbricus terrestris Hemoglobin                                          8419
8420                                 Oxygenation Properties of Lumbricus terrestris Hemoglobin
Oxygenation Properties of Lumbricus terrestris Hemoglobin                                   8421
on ions such as Na+ or Ca...
8422                           Oxygenation Properties of Lumbricus terrestris Hemoglobin
which was converted irreversibly ...
Oxygenation Properties of Lumbricus terrestris Hemoglobin                                              8423
variance of th...
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J. biol. chem. 1986-fushitani-8414-23


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J. biol. chem. 1986-fushitani-8414-23

  1. 1. THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 261, No. 18, Issue of June 25, pp. 8414-8423 1986 0 1986 by The American Society of Biological Chemists, Inc. Printed in L k A . Oxygenation Properties of Hemoglobin from the Earthworm, Lumbricus terrestris EFFECTS OF pH, SALTS, AND TEMPERATURE* (Received for publication, December 16,1985) Kenzo FushitaniSBT, Kiyohiro ImaiB, and Austen F. RiggslI)) From the *Department of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Toyonaka, Osaka 560, Japan, the $Department of Physicochemical Physwlogy, Medical School, Osaka University,Nakanoshima, Osaka 530, Japan, and the TDepartment of Zoology, University of Texas, Austin, Texas 78712 Oxygen equilibrium curves of the extracellular chains of vertebrate hemoglobins (5). The whole moleculehas hemoglobin from Lumbricus terrestris weredeter- been reconstituted from dissociation products and theshape mined under a variety of conditions. These data were has been compared with that of the native molecule by scan- characterized by (i)a rather small free energy of coop- ning transmission electron microscopy (6). Oxygen-binding erativity (1.6-2.8 kcal/mol), (ii) a large and strongly properties of L. terrestris hemoglobin have been studied by pH-dependent Hill coefficient with a maximum value several investigators (7-9). Giardina et al. (10) measured of 7 9 (iii)a high sensitivity of the upper asymptote of ., oxygen binding by earthworm’ hemoglobin under a variety of the Hill plot to pH, and (iv) a maximum association conditions and showed that the Hill coefficient, n, depends constant as large as that of the free subunit of human # I strongly on pH and hasa maximum value of 4 at pH 7.8; the hemoglobin A. oxygen affinity varies between 0.4-7 mm Hg between pH 5.5 The effects of LiCl, KCl, NaCl, BaCl,, CaC12, SrC12, and 10. Vinogradov et al. (11)have reported similar values for and MgClz on the oxygen equilibrium were measured. Cations, not C1-, were found to control oxygen binding. hemoglobin from L. terrestris. Divalent cations have a larger effect on oxygen affinity Recently, Weber (12) showed that cations control the oxy- than monovalent cations, and their effectiveness de- gen affinity of the extracellular hemoglobin of Arenicolu mar- creased in the order listed above within each valence ina. Addition of cations such asNa+ or Mg2+ enhanced coop- class. These specific effects depend in part on ionic erativity and raised the oxygen affinity by binding to hemo- radiusandcannot be explainedinterms of ionic globin at high levels of oxygenation. He also showed that strength.Thedataindicatethatthe oxygenation- protons lower the oxygen affinity by preferential binding to linked binding of a Ca2+ ion is accompanied by the hemoglobin a t high saturation levels. The mechanisms of release of two protons; the binding of a Na+ ion is cationic and protonic interaction in theseextracellular hemo- associated with the release of one proton. These find- globins must be quite different from those in human hemo- ings indicatethat theoxygenation-linked cation-bind- globin A, where anions and protons decrease the oxygen ing site contains two acid groups that do not readily affinity by preferential binding mainly to molecules in the dissociate their protons except when replaced by cat- low affinity state (13, 14). The important study by Santucci ions. et al. (15) has shown for hemoglobin of Octolasium complun- Incubation at either pH 6.2 or 8 9 had no effect on . atum that oxygen binding becomes independent of pH be- subsequent measurements of oxygen equilibria at pH tween pH 7 and 8.5 at sufficiently low cation concentrations. 7.8. The apparent heat oxygenation was found to be of The pH dependence of cooperativity and cationic control of -1 1.8, - . ,and - . kcal/mol at pH 9 0 7 4 and 6.6, 73 93 .,., oxygen affinity has also been found in several other extracel- respectively. These differences indicate that proton- lular hemoglobins of annelids (16-23). binding processes contribute to the heat of oxygena- In the present study we have measured oxygen binding tion. between 1 and 99% saturation with high precision under a wide variety of conditions which includes different kinds and concentrations of salts, changes inpH,temperature,and The extracellular hemoglobin of the earthworm, Lumbricus protein concentration. terrestris, consists of 12 subunits, arranged as two superim- posed hexagonal disks (3), about 30 nm in diameter and 20 EXPERIMENTALPROCEDURES nm high (4)with a molecular weight of 3-4 X lo6. Although Preparation of Hemoglobin-Earthworms, originally obtained in its chain composition and the spatial arrangement of the Ontario, were purchased from the Wholesale Bait Co., Hamilton, OH chains have not yet been determined, one of the polypeptide 45015. They were cut with scissors at a position just anterior to their chains has been sequenced and shown to be homologous to hearts and bled into CO-saturated 0.1 M sodium phosphate buffer (pH 7.0) containing 3 m phenylmethylsulfonyl fluoride as a protease M * This work was supported by National Science Foundation Grants inhibitor. The crude solution was centrifuged to remove cellular PCM 8202760 and DMB-8502857, Welch Foundation Grant F-213, matter. The hemoglobin was precipitated by adding polyethylene and National Institutes of Health Grant GM28410. A preliminary glycol (8,000 average molecular weight, Sigma) to a concentration of account of some of this work has been presented (1,2). Thecosts of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- Originally identified in 1975 as Lumbricus sp. (10) and recently tisement” in accordance with 18U.S.C. Section 1734 solely to indicate reidentified (1983) asOctohium complnmtum (6) although the this fact. worms used by Giardina et al. (10) were referred to as L. terrestris in 11 To whom reprint requests should be addressed. 1984 (15). 8414
  2. 2. Oxygenation Properties of Lumbricus terrestris Hemoglobin 8415 10%.The precipitate was redissolved in 0.05 M Tris-HC1, pH 8.0, and Analysis-Oxygenation data were expressed in terms of the Hill twice pelleted a t 40,000 rpm for 2 h. Pellets were resuspended in this plot (log(Y/(l- Y ) )uersus log P ) where Y is fractional saturation of CO-saturated buffer and stored in liquid nitrogen. For oxygenation the hemoglobin with oxygen and P is the oxygen pressure in mm Hg. experiments, an aliquot of the frozen hemoglobin sample was thawed, Overall oxygen affinity and cooperativity were characterized in terms centrifuged a t low speed, and subjected to gel chromatography on a of oxygen pressure a t half-saturation (PW) maximum slope of the and Sepharose CL-GB column equilibrated with 0.1 M sodium phosphate Hill plot (hex) or slope of the Hill plot at half-saturation (nw). buffer (pH 7.7) containing 0.1 M NaCl saturated with CO. Less than Cooperativity was also expressed in terms of a free energy change 5% of the total hemoglobin was found to be dissociated into %z defined as AG, = R T ln(K,,,/KJ where K,,, and Kl are estimated subunits during the freeze-thaw treatment on the basis of gel chro- association constants for the last and first oxygens bound to hemo- matography. The eluted fractions were concentrated to about 3.2% globin. Values of n were determined by plotting values of n against , w/vby ultrafiltration (Toyo Roshi apparatus, UM-10 membrane). logp. Here, n is the slope of the line connecting two adjacent points The concentrated hemoglobin solutions were dialyzed against 0.05 M on the Hill plot. The apparentassociation constants for the binding Tris-HC1 (pH 7.0 a t 25 "C) containing 0.1 M NaCl and stored on ice of the first and last oxygens to hemoglobin were estimated approxi- as the CO form. mately with an m-step Adair's oxygenation scheme (28), Oxygenation Measurements-Oxygen equilibria were measured with an improved version of an automatic oxygenation apparatus (24) log(Y/(l - Y ) )= log P + log Kl at P o -+ (1) without the use of an enzymatic reducing system. Deoxygenation log(Y/(l - Y ) )= log P + log K,,, at P -+ m (2) and/or reoxygenation data were acquired in real time by a model PDP-ll/vOB microcomputer (Digital Equipment Corp., Maynard, where Kl and K,,, are apparent association equilibrium constants for MA) and were stored on floppy disks. The absorbance value corre- the first and lastoxygens. sponding to 100%saturation with oxygen was obtained by extrapo- The magnitude of the Bohr effect was estimated by the following lating a &4 versus 1/P plot to 1/P = 0 where A.4 is the absorbance expression (29). change upon oxygenation and P is the partial pressure of oxygen (13). The buffer was 0.05 M BisTris/propane' over the range pH 6.2-9.0. h = -Alog Pw/ApH (3) The pH adjusted with concentrated HCl at the was same temperatures Here, h is the number of protons released by Hb/oxygen bound. as those used in the oxygenation experiments. The hemoglobin con- Similarly the magnitude of the effect of a given ion on oxygenation centration was 60 F M on a heme basis, unless otherwise stated. The is expressed by bound CO was removed from the hemoglobin by light while the sample was flushed with pure 0 in a rotating flask immersed in ice ' I = +Alog PW/Alog[ion] (4) water just prior to the measurements. Reproducibility of the Oxygenation Curve-Reproducibility of the where I is the number of ions released by Hb/oxygen bound. oxygen equilibria is affected by two factors: the stability of the The apparent enthalpy of oxygenation, A H , was calculated from automatic oxygenation apparatus and that the hemoglobin sample. of the slope of the plot of log PW versus 1/T between temperatures of Careful inspection of the data showed that oxygenation curves meas- 283 and 303 K. ured under the same conditions within a few days of one another The Monod-Wyman-Changeux (MWC) allosteric model (30) de- could be superimposed over the whole saturation range between 1 scribes the oxygenation of hemoglobin as, and 99% with a variance of 1-2% of P W and n - values where Pw is , the oxygen pressure a t half-saturation and n is the maximum value , - Y= + L K T P ( ~ KTP)""' + + K ~ p ( 1 KRP)""' (5) of the Hill coefficient. Therefore, the reproducibility of the oxygena- L(1 + KTP)" + ( 1 + KRP)'" tion curve was found to depend mainly on irreversible alteration of where rn is the number of interacting binding sites for oxygen, KT the hemoglobin sample during storage such as autoxidation to a and K are theintrinsic association equilibrium constants for oxygen R hemichrome. The PW value dropped by 3%, andn decreased from - binding to the T state and the R state, respectively, and L is the 5.0 to 4.8 at pH 8.9 after 3 weeks. Purified CO-hemoglobin samples allosteric constant. Recently, Decker et al. (31) introduced a conven- gave a variance of about 6% for PW values and 7% for n.-= values ient graphic method for analyzing oxygenation data on the basis of after storage on ice for 3 months. the MWC model. Imai and Yoshikawa (28) modified their formula Estimation of Methemoglobin Content-No complete set of absorp- slightly, as follows. tion coefficients for estimating methemoglobin content over the pH range of 6.2-8.9 is available for this hemoglobin. We failed to deter- log Z = ( m - 1) log X + log L (6) mine the coefficients for methemoglobin because, as Ascoli et al. (25) reported, the spectrum of the L. terrestris hemoglobin changes upon Here, 2 = (KR - Q ) / ( Q - KT), Q = Y/((l - Y)P), and X = (1 + oxidation from that of typical aquomethemoglobin to that of hemi- + K T P ) / ( ~ KRP). Z is the ratio of the difference in affinity between chrome. The millimolar extinction coefficient for oxyhemoglobin at hemoglobin in the R state and hemoglobin a t a given value of P to 576 nm, obtained by the pyridine hemochromogen method, was 16.4, that between hemoglobin at the given value of P and the T state. X which is close to the value of 14.6 a t 577 nm for the oxy form of is the ratio of the binding polynomial for oxygenation of the T state human HbA (26). We used the value of34.4 for the millimolar to that for oxygenation of the R state. When KT and KR values are extinction coefficient of the pyridine hemochromogen a t 557 nm. already known, log Z can be plotted against log X,yielding a straight Methemoglobin content was estimated by using spectra taken before line. The values of m and log L can then be determined from the and after oxygenation measurements. The ratio of absorbance a t a slope and the intercept on the ordinate at log X = 0. In the present trough near 505 nm to that ata peak at 576 nm provides an index of study, we used Kl andK,,, for KTand KR,respectively. methemoglobin. The value of this index for hemoglobin solutions We also attempted to estimate the number of interacting binding between pH 7.0-7.8 before measurements was 2.99 0.03 S.D. (26 sites, m,by using Kegeles' expression (321, m = n,-/y-, derived measurements). The index for human HbA obtained under similar from the MWC model. However, the slope is too steep to permit conditions was 2.99 f 0.06 S.D. (10 measurements). The value of2.99 accurate values to be obtained. for human HbA corresponded to 2.8% f 1.1 S.D. methemoglobin (27). We estimated that 2-4% methemoglobin was present in the RESULTS purified stock solutions of L. terrestris hemoglobin by these approxi- mate criteria. Methemoglobin content after oxygen equilibrium meas- Oxygen-binding Properties: Effectof p H and Calcium-Hill urements was usually estimated to be 6-8% and never more than plots of oxygen equilibria of L. terrestris hemoglobin are given 10% except for three experiments carried out atheme concentrations in Fig. 1. Measurements were made between pH 6.2 and 8.9 under 20 pM (see Fig. 7). Estimation of MetHb content at other pH using 0.05 M BisTris/propane with and without different values was similar. The index decreased to 2.90 f 0.03 S.D. before additional salts. Values of Pw and n obtained from these - measurement 3 months afterpurification. curves are listed in Table I together with other parameters and areplotted against pH in Fig. 2. Fig. 2 also includes data 'The abbreviations used are: BisTris, 2-[bis(2-hydroxyethyl)- from human hemoglobin A (34) for comparison. The datafor MetHb, methemoglo- amino]-2-(hydroxymethyl)-propane-1,3-diol; L. terrestris hemoglobin in Figs. 1 and 2 have four major bin; MWC, Monod-Wyman-Changeux. features as described in the following paragraphs.
  3. 3. 8416 Oxygenation Properties of Lumbricus terrestris Hemoglobin log P FIG.1. Hill plots of oxygen binding by hemoglobin of L terrestris at different pH values and salt . concentrations. Symbols: Y,fractional saturation of hemoglobin with oxygen; P, partial pressure of oxygen in mm Hg. Conditions: 50 mM BisTris/propane, 3 m NaCl, 25 "C, with various amounts of additional salts; M hemoglobin concentration, 60 pM heme. A, no additional salt. pH from the left to right: 8.56,8.13, 7.75, 7.36, 7.00, 6.58, 6.23. B, additional salt, 0.1 M NaCl. pH from the left to right: 8.86, 8.52, 8.10,7.75, 7.35, 6.95, 6.55, 6.16. C, additional salt, 0.1 M NaC1,25 mM CaC12. pH from the left to right: 8.88,8.48,8.10, 7.73,7.36,6.98,6.58, 6.20. The solid straight lines with a slope of unity indicate the lowest lower and highest upper asymptote of the Hill plots. The lowest lowerasymptote was obtained from cunres in A, the highest upper asymptote from C. Their intercepts on the ordinate at log P = 0 give log K L =i -1.6 (KL 0.024 mm Hg") and log KH = 0.58 (KH 3.8 mm Hg-'), = = where K L and KH are the lowest and highest values, covered by the present experiments, corresponding to the association equilibrium constant for the low oxygen affinity state (thelowest first Adair constant, KJ and that for the high oxygen affinity (the highest last Adair constant, Km), respectively. The asymptotes for free B subunit and hemoglobin M Milwaukee corresponding to 4 and 0.004 mm Hg", respectively, are also shown for comparison (33). 1)The shape of the oxygen equilibrium curve varies greatly ative binding of oxygen are smaller than those of human HbA with pH. In the absence of calcium, the value of kx rises (33), although the values of the Hill coefficient are much from 2.5-3.0 at pH6.2-6.6 to a maximum of 6.5-7.2 near pH larger (Fig. 2and Table I). Thelowest valuefor Kl, 0.024 mm 8.1 and decreases to 5 at pH 8.9. The value of nmlu rises to a Hg-l, was obtained in the absence of added salt (Fig. l )A. maximum value of 7.9 at pH7.5 in the presence of calcium; The highest value for K,, 3.8 mm Hg-l, was obtained in the values of n at both extremes of pH remain unchanged. - presence of 0.1 M NaC1, 25 m CaClz (Fig. lc, Table I). The M 2) Salts increase overalloxygen affinity. This effect in- asymptotes corresponding to these values are shown in Fig. creases with pH and is more pronounced with CaClz than 1. Values of free energy of cooperativity (AG,) are listed in NaC1. The pH of the maximum Bohr effect is decreased by Table I. The pH dependence ofAG, is similar to that of hax salts. The maximum valuesof the Bohr coefficient (Alog Ps0/ (Fig. 2).The maximum AG, value, 2900 cal/mol, was obtained ApH) are -0.35 (pH 8.3-8.5), -0.53 (pH 8.6-8.9), and -0.77 in the presence of 0.1 M NaCl, pH 8.52. This value is about (pH 7.4) in the presence of no additional salt, 0.1 M NaCl, 80% of the maximum value,3640 cal/mol, for human HbA in and 0.1 M NaC1, 25 m CaC12,respectively. M 0.1 M Cl-, 2 m 2,3-diphosphoglycerate,pH 7.4 (33). M 3) The two asymptotes of the Hill plot depend on pH Effect of Different Salts on Oxygenation-Fig. 4 and Table differently. The values of Kl and K , calculated from the I1 show the effects of chloride salts of Li+, Na+, K+, Mf, extrapolated asymptotes are listed in Table I. Their pH de- Ca2+,and of mixtures at pH 7.36, 25 "C. Monovalent salts pendence is given in Fig. 3. The value of Kl is about 0.024 have almost no effect on nmaror log Ps0 up to 0.1 M, and mmHg" between pH 6.2 and 8.2 in the absence of added changes inthe Hill coefficient are negligible up to 1 M. salt. However, the value of K , increases from 0.43 mm Hg-l However, the oxygen affinity doubles between concentra- salt at pH 6.2 to 1.9 mm Hg-I at pH 8.6. NaCl and CaClz both tions of 0.1 and 1M.The effect of divalent salts is pronounced enhance the pH dependence of K,. In the presence of CaCh at much lower concentrations; the effect of 0.1 M CaC1, is (Table I, C), the value of K, reaches a plateau near pH 8 similar to thatof 1M NaC1. The combination of Ca2+and 0.1 corresponding to a value of3.8mmHg". The value of Kl M NaCl causes a concentration dependence of log Pm similar increases only slightly with pH in thepresence of Ca2+. to that caused byCa" alone, whereas ha= a quite shows 4) The overall free energychanges associated with cooper- different dependence on CaClz compared NaC1. with
  4. 4. Oxygenation Properties of Lumbricus terrestris Hemoglobin 8417 I I I TABLE I 9 i Values of oxygenation parameters for hemoglobin of L. terrestris obtained under differentconditions of salt and pH In 0 0 M BisTris/propane/HCl buffer, at 25 "C; hemoglobin con- .5 centration 60 PM on a heme basis. A, no additional salt; B, in the c E presence of added 0.1 M NaCI; C, in the presence of added 25 m M CaCL 0.1 M NaCl. ----"""" A 8.56 mm Hg 5.9 6.3 5.4 eallmol 2400 11.2 71 8.9 ' TM T 8.13 8.2 6.5 5.6 2400 12.4 72 9.0 7.75 10.3 6.0 5.2 2300 11.6 72 8.3 7.36 12.7 6.6 5.1 2300 1. 11 70 9.4 7.00 15.2 4.1 3.6 2200 8.5 67 6.1 6.58 16.4 3.1 3.0 1700 6.5 75 4.1 6.23 17.1 2.5 2.3 1700 5.4 63 4.0 B 8.86 2.7 5.0 4.9 2300 9.9 46 10.9 8.52 4.1 6.9 6.6 2900 10.8 61 11.3 8.10 6.3 7.5 6.5 2800 10.8 69 10.9 7.75 9.0 7.2 5.6 2600 10.8 74 9.7 7.35 12.1 6.2 5.3 2400 11.1 73 8.5 6.95 15.4 4.5 3.5 2100 8.4 66 6.8 6.55 17.0 3.0 2.5 1700 6.5 67 4.5 FIG. 2. The effect of pH on overall oxygen affinity (log P) , 6.16 17.3 2.7 2.2 1700 5.1 72 3.8 and cooperativity (%=) at 25 OC. Symbols: Pw, partial oxygen pressure at half-saturation; hx, maximal slope of the Hill plot. C Values of these parameters were obtained from the Hill plots in Fig. 8.88 1.6 4.9 4.9 2500 8.0 57 8.6 1.A, no salt added; 0 , O . l M NaCI; 0 , 2 5 m CaC&with 0.1 M NaC1. M 8.48 1.9 5.8 5.5 2600 9.4 56 10.4 Broken lines show a plot of log P , and for human HbA in 0.1 M 8.10 2.6 6.8 6.4 2800 10.5 61 11.1 NaCl(34). P,,, is the median oxygen pressure. 7.73 3.9 7.9 6.9 2800 12.4 67 11.8 I ' 1 I ( 7.36 6.9 7.7 7.1 2600 11.9 67 11.5 6.98 11.6 5.1 4.2 2100 9.7 70 7.3 6.58 15.5 3.1 2.7 1900 6.2 71 4.4 6.20 17.2 2.6 2.3 1700 5.7 70 3.7 a Oxygen pressure at half-saturation. * Maximum slope in Hill plot. Slope at half-saturation in Hill plot. Free energy of interaction; AG, = RTln(Km/Kl). e Estimated number of interacting sites based on the linearized plot (Equation 6). f Oxygen saturation giving a n - ,. Estimated interacting sites based on Kegeles' analysis. Oxygenation in the presence of 3 m NaC1, 25 m CaCli M M -2.oL " . (Fig. 5) shows that CaClz increases the oxygen affinity and 8 I cooperativity by shifting the upper asymptote to the left. 60 . 7.O 80 . 9.0 PH Although 25 m CaClz has no effect on the lower asymptote, M 125 m CaClz causes a significant shift to the left which is M FIG.3. The effect of pH on K1and K. Kl and K,,, the , are association equilibrium constants for the first and last oxygens to be associated with a decrease in ha. Similar effects were ob- bound. Values of these parameters were obtained from the Hill plots served with MgClz. in Fig. 1. A, no salt added, 0,O.l M NaCI; 0 , 2 5 mM CaClz with 0.1 The effect of 25 m S?+ and Ba" was also examined at M M NaCI; 0, estimated values of Kl andK, obtained by extrapolation pH 7.37. Positions of the lower asymptote of the oxygenation to zero MetHb of the data obtained with partidy oxidized hemoglobin curves were similar to those for Ca" and M$+ whereas the (see t x ) et. position of the upper asymptote differed. The log Pmvalues were: 0.98,0.86, 0.80, and 0.74 for M$+, Sr2+,Ca", and Ba2+, the Hill coefficient by measuring oxygen binding by hemoglo- respectively. Thus, Ba2+with the largest ionic radius was the bin which had been partially oxidized with potassium ferri- most effective and M$+, the smallest ion, had the least effect cyanide at pH 7.3 (Fig. 6). The ferricyanide was assumed to among the divalent cations so far examined, and Sr2+ and react completely with heme. Methemoglobin content up to ea2+with intermediate ionic radii had effects between those 30% had no signifkant effect on the P m value which was of Ba" and M$+. Although the difference between the log 11.2-11.8mm Hg (average, 11.5mm Hg k 0.2 S.D.). The P m values for Sr2+ and Ca" or Ca" and Ba2+are only 0 0 .6 value increased slightly at 50% methemoglobin. However the this is 15% in the value and appears to be well beyond the P value decreased with an increase in methemoglobin (6% estimated experimental error. Inositol hexaphosphate had no decrease with 10% methemoglobin). We estimated values of effect on oxygenation; the data obtained with 2 m inositol M both Kl and K , at 0% methemoglobin byextrapolating plots hexaphosphate at pH 7.41 could be superimposed on those of log Kl or log K , against MetHb content. The extrapolated obtained in itsabsence. values are -1.7 and 0.13 for log K1 and log K,, respectively Effect of Partial Oxidation-We examined the effectof (Fig. 3). These results indicate that Kl and K,,, were not partial formation of methemoglobin on oxygen affinity and affected significantly by the methemoglobin present under
  5. 5. 8418 c 0 :m I T Oxygenation Properties of Lumbricus terrestris Hemoglobin T cg ~~ I 25°C Q! - 0 0 0'50 10 30 50 % Meihernoglobin Salt Concentration Hb Concentration (pM heme) FIG. 4. Effect of salts on overall oxygen affinity (log Pso) FIG. 6 (left). Effect of partial oxidation on overall oxygen and cooperativity (n-). Symbols: PSO, partial pressure of oxygen affinity (log Ps0)and cooperativity (ram-). Oxygenation curves at half-saturation;n- maximum slope of Hill plot. Hemoglobin ,, the were measured i 0.05 M BisTris/propane/HCl, 0.1 M NaCl, pH7.30 n concentration, 60p~ on a heme basis, "C.A, effect of monovalent 25 at 25 "C. Hemoglobin concentration, 60 ptM on a heme basis. The cations: 0, LiCl (pH 7.33-7.36);0,NaCl (pH 7.38-7.39); A, KC1 (pH abscissa showsthe percentage of methemoglobin. 7.36-7.40). B, effect of divalent cations:0, MgClz in the presence of FIG. 7 (right). Effect of hemoglobin concentration on overall 0.1 M NaCl (pH 7.34-7.38); 0, CaClz in the presence of 0.1 M NaCl oxygen affinity (log P60)and cooperativity(k-). Oxygenation (pH 7.36-7.38); A, CaC12 only (pH 7.36-7.40). Broken lines show curvesweremeasured i 0.05 M BisTris/propane/HClbuffer,pH n effect of monovalentsalts; data from A included for comparison. 7.38-7.39, at a concentration of hemoglobin between and 600 p~ 0.6 on a hemebasis. 3t 1 TABLE I1 Effect of salts on overall oxygen affinity and number cations taken of up uponoxygen binding In 0.05 M BisTris/propane/HClbuffer at 25"C; pH7.34-7.40; hemoglobin concentration,60 NM heme. Cation Salt" bound/O. mM 0.75 580 Li+ 940 Na' 0.52 K+ 0.45 820 + M$+ 0.1 M Na+ 110 0.33 Ca2+ 0.37 27 0 + Ca2+ 0.1 M Na+ 0.37 41 -3- * Salt concentration neededt o double overall oxygenaffinity. - 1 0 1 2 3 log P not required to explain the very small shifts. FIG. 5. Effect of calcium chloride on oxygen binding by Reversibility ofOxygen Equilibria with Changes of pH- hemoglobin of L terrestris in terms of the Hill plot. Buffer: . 0.05 M BisTris/propane/HCl and 3 M NaCl (pH 7.36-7.40) at 25 "C. m Giardina et al. (10) reported that the shape of the oxygen Hemoglobin concentration, 60 W M (heme basis). P, oxygen pressure, equilibrium curve of hemoglobin incubated at either pH 6.0 mmHg; Y , fractional oxygen saturation. Ca2+ concentration: from or 10 for 1 h is modified irreversibly. Several workers have the left to right, 125, 25,0 mM. reported similar phenomena for other extracellular annelid hemoglobins (35-37). We have re-examined this property with the conditions used. L. terrestris hemoglobin. Oxyhemoglobin solutions at pH6.2, Effect of Hemoglobin Concentration-Fig. 7 shows the effect 7.8, and 8.9 were prepared as described under "Experimental of increasing the hemoglobin concentration from 0.6 to 600 Procedures." Each solution was incubated for 1 h at 25 "C pM at pH 7.57 in the presence of 0.1 M NaC1. Values of log and passed through a Sephadex G-25 column (pH 7.8) so that P50 nmax not of high accuracy at concentrations of 2 and are the three samples had the same fiial pH (Fig. 8, Table 111). p~ or lower because of significant autoxidation. The 100-fold The plots at the in Fig. 8 show the Hill plots for a control left decrease in concentration from 600 to 6 PM is associated with experiment, where the curves for the hemoglobin samples an increase in Pw of about 7% and a decrease in nmarfrom 6 untreated and treated(i.e. incubated and passed through the to 5. The upper and lower asymptotes of the Hill plots shift column) at pH 7.8 are superimposed. Likewise, the plots at only slightly upon the dilution of hemoglobin sample. The the right in Fig. 8 show five curves. Two of these show data small changes in log PSoand at or below 20 PM heme are obtained at pH 6,2 or 8.9 and not brought back to pH 7.8. very closely correlated with methemoglobin formation; the Three curves show data on samples treated at pH6.2, 7.8, or P0 'value is linearly related to the MetHb index with. a 5 8.9 and brought back to pH 7.8. Over a range of 1-99% correlation coefficient of 0.97. The MetHb index is invariant saturation, their agreement is excellent. It was found that between 60 and 600 pM heme. Although a small degree of addition of 25 m CaClz to a solution incubated at pH 8.9 M subunit dissociation may accompany dilution, dissociation is improved the reversibility even further for pH changes from
  6. 6. Oxygenation Properties of Lumbricus terrestris Hemoglobin 8419 I I I - n 0 8 *A pH 8 9-7.8 . 0 .E FIG. 8. Test of the reproducibility of oxygenation after changing the buffer. Hemoglobin solutions incubated for 1 h at each pH (untreated; shown with open symbols) were brought back to the same pH 7.8 with Sephadex G-25 (treated; shown with solid symbols). Left two curves are shown for control pur- poses. 0 -2 1 -I 0 I t I I I I -I 0 I 2 -I 0 I 2 log P TABLE I11 TABLE IV Reversibility of oxygenation parameters upon changing pH of Apparent heat of oxygenation (kcal/mol) hemoglobin solution In 50 m BisTris/propane/HCl buffer with 0.1 M NaC1. Hemoglo- M In 0.05 M BisTris/propane/HCl buffer with 0.1 M NaCl at 25 "C. bin concentration, 60 pM heme. Heats of oxgen binding at oxygen Hemoglobin concentration, 60 pM heme. Buffer was changed with saturations of 50%. Values were corrected for heat of solution of Sephadex G-25. P50, oxygen pressure at half-saturation; n-, maxi- oxygen (3 kcal/mol). mum slope in Hill plot. pH L. terrestris Human H b Condition" PO S n, - MetHb indexb 9.0-9.1 -11.8 -15.3" 7.8 (untreated)" 6.7 7.64 2.85 2.99 -9.3 7.4 -7.5 2.67 72.81 . 8 .h7 6.0 7.59 -11.2 6.5-6.6 -9.3 5.9 7.83 6.2-7.8 In 50 mM BisTris or Tris/HCl buffer with 0.1 M NaC1, at pH6.5, 4 2.80 5.9 7.87 7.4, 9.1 (27). 8.9-7.8' 7.83 5.1 2.42 2.95 Left column shows starting conditions. Right column shows con- pH dependence of n,,, are qualitatively in agreement with ditions after change (see legend for Fig. 8). results obtained by Giardina et al. (lo), Santucci et al. ( E ) , Ratio of absorption at minimum value near 505 nm to that at the and Vinogradov et al. (ll), a peak in oxy form. Left, before measurement; right, after measure- although our n values are consist- ment. ently higher evenwhenexpressed as nsOrather than n- No buffer change with Sephadex G-25. (Table I). Qualitatively similar properties have beenreported Incubated at pH 8.9 with 25 m CaC12. M for extracellular hemoglobins from other annelids, both po- e Incubated at pH 8.9 without 25 m CaC12. M lychaetes and oligochaetes: Amphitrite ornata (20), Lumbri- nereis tertraura (19), Pheretima hilgedorfi (22), and Eiseniu 8.9 to 7.8. The numerical data for these experiments (Table foetida (23). 111) show excellent reversibility with respect to pHchange. This pH dependence has been suggested to result from a Effect of Temperature on Oxygenation-Oxygen equilibrium greater sensitivity of the upper asymptote to pH than the curves were determined at 10,15, 20,25, and 30 "C at pH 6.6, lower one (20). Recently, Weber (12) clearly showed for A. 7.4, and 9.0 in the presence of 0.1 M NaC1, but satisfactory marina hemoglobin that theupper asymptote in theHill plot data at pH 9.0 and 30 "C could not be obtained because of shifted to the left with an increase in pH but that the lower significant autoxidation. The dependence of oxygenation of asymptote hardly moved. He suggested that theoxygen affin- L. terrestris hemoglobin on temperature is similar in extent ity of the high affinity states depends on cation and proton to that for human HbA (38). Overall heat of oxygenation binding. Our data (see Fig. 3) for L. terrestris hemoglobin are (AI&) was obtained by plotting log Pm against 1/T. The completely consistent with this picture. The upper asymptote values are listed in Table IV, together with data for human is more sensitive to pH than the lower one although the lower HbA (27). asymptote does shift slightly above pH 8.0 in the absence of salts andgradually shifts to theright in thepresence of 0.1 M DISCUSSION NaCl and 25 m CaClz abovepH 7.0. M Oxygenation Characterization of L. terrestris Hemoglobin- The data in Fig. 3 may be interpreted in terms of at least Oxygenation parameters for L. terrestris hemoglobin in the two high affinity statesin L. terrestris hemoglobin.One present work are compared in Table V with those for the appears at low pH independent of the presence of salts (KmL same hemoglobin obtained by other workers, for hemoglobin = 0.43 mm Hg-') and the other at high pH and/or high salt from two related earthworms, for chlorocruorin from Potam- concentrations (K," = 3 8 mm Hg-l, consequently equal to . ilk leptochaeta, and for human HbA. Our data on the strong KH). Free energy differences between KL and KmL,and K,"
  7. 7. 8420 Oxygenation Properties of Lumbricus terrestris Hemoglobin TABLE V Oxygenationparameters for extracellular hemglobin and chloroeruorin Temperature Source" pH P, n Bohr effect AHb Reference oc L. terrestris 25 6.2-9.0 1.63-17.3 2.5-7.9 -0.35-0.77 -7.5 to -11.8 7 7.3 2 -8.0 20 7.3 a (3.4)' 10 7.12 3.5 (3.0) -0.25 10 7.21 4.8 (2.3) 15 7.10 2.88 5.21 -0.4 15 7.44 3.89 5.20 15 7.10 4.98 5.13 25 7.70 5.28 5.41 -0.4 -10.2 25 7.44 6.78 5.30 -9.1 25 7.10 9.20 5.11 -10.6 22 6-9 (2.7-15.8) (1.8-5.0) (-0.54) 0. complnnatom 20 5.4-10.0 (2.2-6.9) (1.6-4.2) (-0.64) -6 to -13.7 E. foetida 20 5.1-9.4 (0.59-4.4) (1.8-3.5) -0.44 P.leptochaeta 25 6.2-9.2 11-420 1.14-5.82 -0.98 -3.9 Human HbA 25 6.0-9.0 161 .-6 2.53-2.98 -0.53 -11.2 to -15.3 'P. leptochaeta is the source of chlorocruorin; the othersare thoseof hemoglobin. Values have been corrected for the heat of solution of oxygen, 3.0 kcal/mol, except for those from Ref. 9. - Values in parentheses areobtained bv reading or recalculating values from published data. See Footn>te 1. and KmH 1700 and 1300 cal/mol, respectively. The change are a strongereffect than M$+ in E. foetida (23) and L. terrestris of free energy of cooperativity with pH (Table I) is essentially (present data), but in ornata (20) the opposite is true. A. parallel to that of n- under three sets of conditions. Thus, Similar effects of salt concentration on log P 0 and 5 the pHdependence of the shape of the oxygen-binding curve (Fig. 4B) have been found in several other extracellular hemo- between pH 6.2-9.0 results from the relative movements of globins (11, 15, 18, 23). One interpretation is as follows: (i) the lower and upper asymptotes. The increase in at low cations bind to the liganded form of hemoglobin more than pH appears to be due exclusively to an increase in the K,,, to the unliganded form at relatively low salt concentrations, value with constant Kl) and the decrease in n,, at alkaline resulting in a shift of upper asymptote leftward (Fig. 5); (ii) pH is due to a slight increase in K1 with the upper asymptote changes of n- value with cation concentration are due to remaining unchanged. On the same basis, the shift of pH relative movements of both the upper and lower asymptotes. giving the maximum value in thepresence of 0.1 M NaC1,25 The fact that theupper asymptote shiftstoward the left with mM CaClz can be explained by a sharp increase of the K, increased concentration of cations while the lower one re- value to itsmaximum combined with a more gradual increase mains almost unchanged (except at high cation concentra- of Kl (Fig. 3). A similar effect has been observed in chloro- tion) indicates that cations bind to hemoglobin at late stages cruorin (28). of oxygenation. These features are consistent the results with Similar shapes of the pH dependence of n have been - obtained for A. marina hemoglobin (12) and P. leptochaeta reported for several extracellular hemoglobins and chloro- chlorocruorin (28) and seem to be a general characteristic of cruorins from different annelid species (8-12, 16-23, 37, 39- extracellular hemoglobins and chlorocruorins of annelids. 45) where the pHgiving a maximum n varies from pH 7.5 to Magnetic quadrupole relaxation experiments suggest that 9.0. Chiancone et al. (35) reported an exceptional case where Na+ may compete with Ca" for the same site in Lumbricus the shape of pH dependence of the Hill coefficient is concave sp.' hemoglobin (45). The stoichiometry of Ca2+binding was upward as observed for %z subunits from Affinis affiinis(44). estimated to be 0.26-0.31 Caz+/heme. These values are very Effect of Salts-The present experiments show that the close to our values: 0.33 M$+ and 0.37 Ca2+ions taken up per effect of different salts on oxygen binding are specific and oxygen bound (Table 11). Chiancone et al. (20) reported that depend exclusively on the cations. Comparison of data at the 1.6 oxygenation-linked Ca2+ions/heme occur in the pH range same c1- concentrations (Fig. 4B), one set at 50 m CaClz M 7.7-8.5 and suggested that carboxyl groups with abnormal pK and the other at mM NaCl or at the corresponding ionic 100 values may be responsible for the Ca2+-binding site for A. strength of NaCl (150 mM), shows clearly that the increase ornuta hemoglobin. However, it seems rather unlikely that in oxygen affinity iscaused by Ca2+,not by C1-. The data also carboxyl groups themselves would have pK values this high. show that theeffects cannot be explained merely in terms of Makino (46) showed for hemocyanin from Dolabella auricu- changes in ionic strength. The absence of an effect of C1- on laria that H+ and may compete for the same binding site Ca2+ oxygenation issupported by nuclear magnetic quadrupole on the basis of measurements of equilibrium dialysis and H+ relaxation experiments, where C1- binds to both liganded and titration. He also suggested that theCaz+-bindingsite(s) may unliganded forms of the hemoglobin with the same affinity include a histidine residue on the basis of the calculated pK (45). The stronger effect of divalent than monovalent cations value of the protonated site. is probably also true for other extracellular hemoglobins and Our data (lower panel in Fig. 2) suggest that themaximum chlorocruorin (12, 20, 28). Among divalent cations, Ca2+has number of oxygenation-linked protons apparently depends
  8. 8. Oxygenation Properties of Lumbricus terrestris Hemoglobin 8421 on ions such as Na+ or Ca2+ and reciprocally, the protons contains 70 Ca2+relative to 160 iron atoms. The sample was which are released in the presence of Na+ or Ca" depend on dialyzedexhaustively against distilled deionizedwater or pH. Ourobservations are consistent with those of Santucci et against 10 m EDTA, pH 7, followedby dialysis against M a. (15) on the hemoglobin of Octolasium cornplanaturn. We l water. Chiancone et al. (45) suggested that Ca2+acts as a suggest that both sets of data may be explained in follow- the cross-linking agent between two carboxyl groups anchored at ing way.We suggest that certain oxygenation-linkedacid the interface between two "one-twelfth"subunits. The whole groups are present which have highpK values (at least 9.5 or molecule dissociates to %z subunits at alkaline pH, but this higher ). If cations could bind only to thedeprotonated group dissociation is prevented by Ca2+ions (49). The whole mole- with even a low affinity the fraction of molecules with the cules of some hemoglobins dissociate evenneutral pH upon at acid groups dissociated would be greatly increasedat a much removing Ca" ions (20, 50). Onecan, therefore, ask whether lower pH than would otherwisebepossible. Assume the "structural" Ca2+ and "functional" Ca" have the same or following simple sequence illustrative purposes, for different binding sites. Chiancone et al. (20) suggested that structural binding sites differ from functional ones on the basis that a concentration of Ca2+ions only slightly above1 m Ca2+is enough to stabilize the whole structure whereas M where K, is the acid dissociation constant K z is the binding more than 10 mMCa" is necessary to effect a change in the and constant forthe cation. If the apparent dissociation constant in functional properties. It would, therefore, seem to be impor- the presence of cations is given by Ki = ([Hb] + [HbNa]) [H+]/ tant to determine the level of subunit dissociation at which [HbH+]then the apparent pK; will be given by functional alteration by Ca2' can be observed. If L. terrestris pK; = pKl - log(1 + Kz[Na+]) hemoglobin wereto have the same number bound Ca" ions of as T. tubifex hemoglobin, 0.44/heme (48), it would mean that If Na+ = 0.3 M and we assume only a very modest binding deoxygenation would be associated with the dissociation of constant for Na+, say 100, this would be sufficient to lower most of the Ca" since 0.37 Ca2+ ions become boundper the pKl value by 1.5 units. The greater effects of Ca" could oxygen bound or one Ca+ per 3 0 . this were true then at , If be explained by a much larger value of K2. Since the log P ~ o least 84% of the Ca2+would be functional and thepossibility values at high and low pH in the presence of sufficient salt is raised that uniquely structural nonfunctional Ca2+may not differ by about 1.1, the value of the product KlK2would shift exist. The lowest concentration of added Ca" which we have by the same amount because of the linked-function relation- used is 0.2 mM. The estimated amount of endogenous bound ship. One cannot tell on the basis of the present data whether ca2' is no more than 26 pM (0.44 X 60 pM) or 13% of that values of Kl and K, are both oxygenation dependent. This added, an amount that appears to be too small to affect our simple model is probably a considerable oversimplification Bohr proton calculations. but suffices to show that cations can lower the pH at which Weber and Olsen (51) have recently sought to explain the the Bohr effect couldbe observed. Analternative would be to dependence of oxygen binding on cations by invoking a con- assume that the cation binds first and that this results in a cept of "surface pH." They argue that the cations do not exert lowered pK for the acid groups. The net result would be the their effect by binding to specific sites but by altering the same. surface pH. The basis of this conclusion rests on a Gouy- Thedata indicate that approximately 0.77 protons are Chapman planar model with a fixed uniform charge density released and 0.37 Ca2+are taken up per oxygen bound at pH on the surface. However, amino acid sequence and composi- 7.4 almost exactly 2 protons/calcium ion. Similarly, for mon- tion data indicate that the net negative charge results from ovalent ions, 0.52 Na+ ions are taken up and 0.54 protons only a small difference between largenumbers of both posi- released per0 2 bound givinga ratio of 1:l. Although the Bohr tively and negatively charged groups which havevery non- a effect in the presence of Li+ has not been measured, it is uniform distribution. This means that any electrostaticeffects striking that the number of Li+ ions taken up per 0,bound on ion distribution must be extremely local. Although such at pH 7.4 is 0.75, close to thevalue forthe Bohr effect in the effects would give rise to local Debye-Hiickeldistributions of presence of calcium. These results indicate the presence of ions, the concept of surface pH does not appear physically two acid groups.Since a Ca2+ ion may becoordinated with up meaningful. Furthermore, the very high mobility of protons to 8 ligands (47)an attractivepossibility is that two salt links would tend to homogenize this effect. Weber and Olsen (51) of the form, -NH,+ -0OC-, are present. The pK value of the excluded binding at specific sites largely on the basis that assumed -NH,+ wouldbe raised to over 10 because of the choline and Na+ appeared to have the same effect even though influence of the -COO- group. Ionization of two such groups the two cations differ greatlyin size. This could be fortuitous might lead to a binding site for Ca2+ with least four groups. because our at data on four different divalent cations (Ca2+,Sr2+, Ba", M P )show that each has a unique effect which cannot be explained the basis of ionic strength. Rather, the effects on appear to depend at least in parton ionic radius. Monovalent ions (Li+, Na+, K+) also have specific effects; the number -NH; i "OOC bound per O2 bound is closely related to ionic radius: the smallest Li+ is bound to the greatest extent, Na+ is next, The Ca2+ could coordinate with other protein groups such followed bythe largest ion, K'. Although the effects of cations also as the carbonyl group of the peptide bond or other amino acid on annelid hemoglobins must involve electrostatic binding, side chains and/or with water molecules (47). This model this does not excludespecificity.Detailed studies of pH- would explain nicely the observed relationship between the dependent processes in many proteins (52) indicate that a Bohr effect and the binding of both monovalent and divalent primary role is played by electrostatic modifications of pK cations. values and by conformation-dependentchanges in hydrogen The assembly of extracellular hemoglobins fromtheir con- bonding. stituent subunits is well known to depend on Ca2+. Rokosz Reversibility of Oxygenation Properties upon pH and Viogradov (48) reported on the basis of x-ray fluores- Change-Giardina et al. (10) concluded that "native" hemo- cence that thewhole molecule of Tubifex tubifex hemoglobin globin of the earthworm' was in a metastable conformation
  9. 9. 8422 Oxygenation Properties of Lumbricus terrestris Hemoglobin which was converted irreversibly to a more stable state when ent enthalpy of oxygenation in Lumbricus hemoglobin will the Hb solution was brought to neutral pH after incubation also include contributions from proton reactions. Since Bohr at either pH 6.0 or 10 for 1 h. The basis of this conclusion proton release in Lumbricus hemoglobin is coupled to cation was the finding that this treatment appeared to decrease the binding, the enthalpy of cation binding will also be involved. value of the Hill coefficient irreversibly.Several workers (35- Functional Unit-Since the hemoglobin of L. terrestris has 37) have supported the idea of “a metastable state” by observ- about 200 02-binding subunits the task of describing the ing such an irreversible decrease in n. This phenomenon was oxygen equilibrium directly in terms of a 200-step Adair model also believed to be supported by circular dichroism (53) and is clearly hopeless. However,if it could be demonstrated that small-angle neutron-scattering experiments (36).However, cooperative oxygen binding involved only small set of inter- a our results (Fig. 8 and Table 111) show that the reduction in acting groups, the problem would be easier to handle. Several cooperativity upon exposure to acid or alkaline pH is com- attempts havebeenmade to estimate the size of such a pletely reversible. The MetHb index shows that themanipu- functional unit in annelid hemoglobins and chlorocruorins. lation involved in this experiment such as equilibration with Weber (16) reported that at least five 02-binding sites in A. Sephadex G-25 or incubation in dilute solution of HbOz at marina hemoglobin constitute an interacting group. Wyman 25 “C increased the MetHb content compared to that of (29) reported at least 5 interacting sites for Spirographis untreated Hb. In the case of incubation at pH 9:0 without spallanzanii chlorocruorin and Colosimo et al. (59) reported Ca2+, n value decreasedsignificantly, apparently irrevers- the 10 sites for the same chlorocruorin. I a and Yoshikawa (28) mi ibly. Such a drop in the MetHbindex is serious. Fig. 6 shows reported 6 interacting sites for P. leptochaeta chlorocruorin. thatthe presence of MetHb reduces the value buthas All of these estimates have been based on the MWC model little effect on the P, value. We conclude, therefore, that and/or deductions based onthe Hill coefficient. We have also ”irreversible” decrease in n at pH 9.0 without Ca2+, as - attempted to estimate the size of the functional unit on these observed in this experiment, was caused by the formation of bases. A plot of oxygen equilibrium data interms of a linear- MetHb. Presumably the MetHb formation would have bees izedMWC equation (Equation 6) is shown in Fig.9. The even higher at pH 10. Our results are consistent with the value of X in the figure is calculated from the relation, X = report that Ca2+prevents dissociation of the whole molecule (1 + KTP)/(l + KRP).Although the data are nonlinear, the and also protects against autoxidation (48,53-55). maximum slope in the central part was used to provide the Martel et al. (36) reported an irreversible decrease in the values shown in Table I. These data show that the apparent Hill coefficientfor L. terrestris hemoglobin that they ex- number of interacting sites appears to vary between 5 and 12, plained as resulting from mismatched reassembly upon re- depending on pH. Maximum values were obtained between turning the solution to neutral pH. Carbon monoxide was pH 7.8 and 8.2. This corresponds to the pH range which gave apparently not used as a protective agent during hernoglobin the maximum valuesof n. Similar estimates of the number of preparation and storage (56), and Ca2* appears not to have interacting sites were obtained with Kegeles’ method (32) and beenusedwhenhemoglobinwasdialyzed against pH 9.0 are listed in Table I together with y,,. The datasuggest that buffer (36).A significant amount of methemoglobin can form the size of the functional unit depends not only on the pH under these conditions, as described above. We believe, there- but also on the kind and concentration of salt. Although the fore, that theapparently irreversible decrease in the value of calculations give plausible numbers, all of which are very n may well result from the formation of methemoglobin. small compared to the total number of hemes/molecule, they Circular dichroism experiments (53) showed spectral are based on a two-state MWCmodel.Wehaveshown, changes in the Soret region that were attributed to a local however, that a two-state model isnot sufficient for the alteration of the heme environment. This local change, how- description of the data. The nonlinearity of the curves inFig. ever, does not appear to exclude the possibility of MetHb 9 emphasizes this point. An alternative procedure would be formation for the following reasons. Harrington et al. (57) to adopt a completely statistical approach to binding. More reported for L. terrestris hemoglobin that the CD spectrum generally, without recourse to any model of hemoglobin be- has maxima at 412 and 422 nm in theoxy and cyanmet forms, havior, nmax be shown to be proportional to thestatistical can respectively. Ascoli al. (25) reported that theaquomethem- et oglobin form at pH 7.0 irreversibly changed to a hemichrome after bringing the pH to 6.0 or 7.6. This hemichrome may cause a change in the heme environment which would result in a different CD spectrum. Although small-angle neutron- scattering data (36) show a scattering curve different from that of the parent molecule, the difference is difficult to interpret unambiguously. - In contrast to the irreversible changes reported for “Lum- bricus SP.’’~ (IO), Santucci et al. (15) and Chiancone et al. Hb (20) have reported reversible changes in n with pH for the hemoglobins of both 0. complanatum and A. ornata. These findings together with our data suggest that the existence of a metastable state of ferrous hemoglobin is unlikely. Temperature Effect-The variation of A H S O with pH is very similar tothat of human HbA (Table IV). An enthalpy -2t i difference of 3.5 kcal/mol observed at pH 9.0 between L. -2 -I 0 terrestris hemoglobin and human HbA is larger than those at log x pH 6.6 (1.9 kcal/mol) and pH7.4 (1.8kcal/mol). Studies have FIG. 9. Plots of oxygen binding by. terrestris hemoglobin L shown (27, 58) that the intrinsic heat of heme oxygenation according to the linearized MWC equation(Equation 6 in for human hemoglobin is -14.4 to -15.6 kcal/mol of oxygen text). Kl and K,,, (Table I) were used for KT and KB, respectively. bound. Variation in the apparent enthalpy appears to result Buffer: 0.05 M BisTris/propane/HCl, 0 1 M NaC1.pH from left to . entirely from proton and other nonheme ligands. The appar- right: 8.10,7.35,8.86, 6.55.
  10. 10. Oxygenation Properties of Lumbricus terrestris Hemoglobin 8423 variance of the distribution of bound ligand among sites (60, 26. Antonini, E., and Brunori, M. (1971) i Hemoglobin and Myoglo- n 61), and so a high Hill coefficient reflects a highly nonrandom bin in the Reactions with Ligands, North-Holland Publishing distribution of bound ligands among possible sites. At least Co., Amsterdam 27. Imai, K. (1979) J. Mol. Biol. 133,233-247 four intrinsic 02-bindingequilibria must be involved because 28. Imai, K., and Yoshikawa, S. (1985) Eur. J. Biochem. 147, 453- 4 major kinds of subunits with unique 02-bindingcharacter- 463 istics have been isolated (1).One can hope to establish a 29. Wyman, J. (1964) Ado. Protein Chem. 19, 223-286 better basis upon which to describe the oxygen equilibria by 30. Monod, J., Wyman, J., and Changuex, J. P. (1965) J. Mol. Biol. obtaining detailed data on the subunits'in various states of 12,88-118 assembly and so to obtain a description of the whole molecule 31. Decker, I., Sabel, A., Linzen, B., and Van Holde, K. E. (1983) in Life Chemistry Reports (Wood, E. J., ed) Suppl. 1,pp. 251-256, in terms of its 4 major constituent subunits and theirmeas- Harwood AcademicPublishers, London urable interactions. This procedure will also help ascertain 32. Kegeles, G. (1979) FEBS Lett. 103,5-6 whether the minor chains V and VI (Ref. 6) play any impor- 33. Imai, K. (1982) Allosteric Effects in Haemoglobin, pp. 113-114, tant functional or structural role. Cambridge University Press, London 34. Imai, K., Yonetani, T., and Ikeda-Saito, M. (1977) J Mol. Biol. ' ? Acknowledgments-We wish to express our thanks to I. Tyuma Dr. 109,83-97 of the Medical School at Osaka University and Dr. H. Morimoto, 35. 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