Bauer, Christian

Kurtz, Armin

Rollema, H. S.

English

University of Regensburg Publication Server

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 210, No. 1, August, pp. 200-203, 1981 Heterotropic interactions in Monomeric (IS” Chains from Human Hemoglobin ARMIN KURTZ, HARRY S. ROLLEMA, AND CHRISTIAN BAUER hstitut fiir Physiologic, Universitdit Regensinwg, 8.400 Regenaburg, West Germany Received July 30, 1980 The O2 affinity of fiSH chains is lowered by H+, inositol hexaphosphate (IHP), and COz. As the oxygen affinity of gSH monomers (flISH) is lower than that of fiSH tetramers (@dSH), it is possible that IHP and CO2 exert their influence on the Oz affinity of PSH chains by increasing the dissociation constant of +3dsH rather than by a direct effect on the molecule. In order to test for this hypothesis we have measured the 02 affinity of fiSH chains as a function of protein concentration at various concentrations of IHP and inorganic phos- phates in the absence and presence of COz. From these data association constants for the binding of IHP to /31SH and j34SH as well as for the equilibrium 4j31SH = j3dSH were calculated. We found that IHP and CO2 influence the oxygen affinity of flISH. It was furthermore established that inorganic phosphate enhances the stability of PdSH while IHP favors its dissociation in monomers. Recently it has become evident that het- erotropic interactions between oxygen and protons, inositol hexaphosphate (IHP),’ and CO2 are not restricted to the normal hemoglobin tetramer, ~$3~~ but also exist in homotetramers consisting of BSH chains, flssH (l-3). Furthermore, it has been shown that the oxygen affinity of BSH monomers (PlsH) and ASH is different (4, 5) and that it is pH dependent both in BdSH and PISH (5). Moreover, it was established that the dissociation of ,kJSH in 4 PISH is dependent upon pH (5). In view of these results we were interested to see if IHP and CO2 also influence the oxygen affinity of DISH and furthermore wanted to establish if the monomer-tetramer association constant of P SH chains is affected by the polyanion IHP and by inorganic phosphates (Pi). MATERIALS AND METHODS aSH and @ ‘” chains from human hemoglobin were prepared as described previously (2,3). Sperm whale myoglobin was obtained from commercial sources (Serva, Heidelberg) and the heme iron converted from the trivalent to the divalent form as previously described (6). The proportion of oxidized heme groups was 3% in this material. The chains were stored in 1 Abbreviation used: IHP, inositol hexaphosphate. liquid nitrogen until use. Oxygen binding curves were measured at 20°C with heme concentrations ranging between 2 PM and 2 mM. Experiments at low heme concentration (co.5 mM) were performed according to Benesch et al. (‘7) using tonometers with a lo- or 2-mm cuvette fused to it. For the experiments in the presence of CO* a tonometer with a rubber sealed sidearm was used through which the desired volume of CO2 was injected (8). Oxygen binding curves at heme concentration > 0.5 mM were measured with a diffusion chamber (Eschweiler, Kiel) as described by Jelkmann and Bauer (9). Buffers were 0.05 M N,N’- methylenebisacrylamide-Tris in absence and NaHC4 in presence of COz. The pH was kept constant at ‘7.3 within 0.02 pH unit in all experiments. Total [A-] = 0.15 M and temperature 20°C. All buffers contained 0.1 mM EDTA. In the experiments at low-protein con- centrations the oxygen binding curves were obtained from experiments in which deoxygenation was fol- lowed by only one single addition of air in order to avoid excessive heme oxidation during the experi- ment (7). Heme oxidation was found to be enhanced at low heme concentrations. The maximal percentage at the lowest heme concentration studied was 8% at the end of the experiment. For the following analysis it is assumed that the measured Pm is equivalent to the median ligand affinity. This assumption is cor- roborated by the fact that the n value was not sig- nificantly different from unity in our oxygen binding curves. Mathematical procedure. In order to evaluate the concentration dependence of log Pw the following model was used: 0003-9861/81/090200-04$02.00/O 200 Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. HETEROTROPIC INTERACTIONS IN fllSH MONOMERS 201 -6 -5 -4 -3 1% CHb FIG. 1. Plot of log Pa (the partial pressure of 02 at half saturation of the pigment) against log c, the concentration of bSH chains expressed in heme equiv- alents at various concentrations of IHP. Tempera- ture was 2O”C, pH 7.3, and Cl- = 0.15 M. The lines connecting experimental points were calculated as described in the text. P4 SH e 48,s” PISH + 02 = PISH(Od PdSH + i0, F! P&SH(O*)i i = 1, 2, 3, 4 Assuming that oxygen binds noncooperatively to /3aSH the fractional saturation, Y, at a given partial oxygen pressure, p, is given by y= [81KdP + 4L3l%,I~KT.P(1 + KT.PY MU + KMP) + 48Pfi,4U + &PI4 with KM and KT the microscopic association con- stants for oxygen binding to j3iSH and fldsH, respec- tively; 8& the association constant for deoxygenated Bl SH; and w] the concentration of unliganded fllSH chains, i.e., flSH monomers. [8] is given as the root of the following equation: C lot = kW + KM .P) + 4U3PK&U f KYPY, where ctot is the total heme concentration. KM, KT, and @d were obtained from the relationship between the experimental log Pr,,, and the protein concentra- tion using an iterative least-squares procedure. Val- ues for Pm of prsH and BaSH were calculated from KT and KM, respectively. c$ was computed from Z&, Ku, and KT. IHP binding constants for fllSH and @iSH were cal- culated from plots of log Pm against the IHP con- centration for fiiSH and jIISH according to the equa- tions derived by Baldwin (10) and Szabo and Karplus (ll), assuming four oxygen-linked IHP binding sites for fidSH and one for fiiSH. RESULTS In Fig. 1 is shown a plot of log PSo against the PSH concentration for a num- ber of IHP concentrations. It can be seen that the oxygen affinity decreases with decreasing protein, concentration at all IHP concentrations investigated. Experi- ments with aSH chains showed that at pH 7.35 the oxygen affinity of lySH is indepen- dent of protein concentration in a range between 10 and 120 I.LM heme and also in- dependent of the presence of 30 mM IHP, log PSO being -0.42 + 0.01 at 2O’C. Like- wise, PW of sperm whale myoglobin was independent of both protein concentration and IHP concentration (log P,,,: -0.35 f 0.02). A plot of log PSO against IHP concen- tration for pi’ and PdSH is shown in Fig. 2. Here it becomes evident that the oxygen affinity of both filSH and PdSH depends upon the IHP concentration. Figure 3 shows a plot of log PbO against the concent.ration of flSH chains in presence of CO2 @CO, = 100 Torr). It can be seen that over the whole range of protein concentrations in- vestigated the oxygen affinity in the pres- ence of COe is lower than in the absence of COz. In the presence of 15 mM IHP, the addition of COz does not result in a further decrease in oxygen affinity showing that the influence of IHP and COz on the oxygen affinity of PSH chains is not additional. At lop4 M heme for example, the presence of COz and IHP decreases PN by a factor of 1.7 while the sum of the effects should decrease the ligand affinity by a factor of 3.3. Table I shows log Pw values at various heme concentrations in the absence and presence of inorganic phosphate. It can be 0 10 20 30 40 50 &, [IHa.mM FIG. 2. Plot of log Pa against the concentration of IHP for BiSH (upper curve) and fllSH (lower curve). The lines were calculated as described in the text. 202 KURTZ, ROLLEMA, AND BAUER e+3g cl -e FIG. 3. Plot of log Pm against the concentration of gSH chains in the presence and absence of either COn or IHP or both. The line connecting the experimental points at pCOe = 100 Torr and in the absence of IHP was drawn by inspection. The dotted curve was taken from Fig. 1. The line connecting points obtained in the absence of COe and IHP was calculated as de- scribed in the text. seen that at a heme concentration higher than 40 PM the oxygen affinity of PSH chains becomes essentially independent of protein concentration in the presence of 0.15 M Pi. From Table II it can be seen that the apparent association constant of the equilibrium 4plSH G= pdSH in the presence of IHP decreases with increasing IHP con- centration approaching a constant value at high IHP concentrations. DISCUSSION The results obtained in this study clearly show that IHP and COP reduce the oxygen affinity of ,& sH. This, in turn, implies that oxygenation of the plSH causes changes in tertiary structure of the molecule that are large enough to have a distinct influence on the binding sites of IHP and COz. The TABLE I DEPENDENCYOFLOGP~ ONII'i]ANDIC1-] AT VARIOUSCONCENTRATIONSOF~~ CHAINS Log p50 0.05 M cl- Log c 0.15 M cl- O-1 M Pi 0.15 M Pi -3.5 -0.82 -0.80 -0.75 -4.0 -0.75 -0.80 -0.75 -4.4 -0.68 -0.80 -0.75 -5.0 -0.55 -0.73 -0.73 -5.1 -0.53 -0.71 -0.72 effect of IHP on P5,, is significantly stronger in /3i SH than in fldsH. This result is of particular interest as both in isolated cr chains and in sperm whale myoglobin, the Pso was completely independent of pro- tein concentration (lo-120 PM heme) and also of IHP. From Table II it is seen that K1,4reaches a constant value at high IHP concentra- tions. Since -6log K1,&log [IHP] repre- sents the number of IHP molecules re- leased upon tetramer formation, this result means that at saturating IHP concentra- tions the filSH -+ BdSH reaction is not as- sociated with a change in the number of moles IHP bound. So if one assumes one binding site for IHP in DISH, it follows that four IHP molecules are bound per PdSH. Using the data given in Fig. 2 we cal- culated association constants for IHP to deoxygenated (Ku) and oxygenated (Ko) Bl SH and BISH, assuming one oxygen-linked binding site for IHP per fllSH and four equivalent oxygen-linked binding sites for #kSH. The following figures were obtained: K,, = 476 (M-l), K. = 211 (M-l), for PISH KD = 140 (M-l), Ko = 80 (M-l), for j34SH, where KD and KO for @dSH represent the microscopic binding constants. Salahuddin and Bucci is due to the lower The value of KO for fldSH is by two orders salt concentration (0.05 M Cl-) used by of magnitude smaller than the figure re- these authors in their experiments in com- ported by Salahuddin and Bucci (12), who parison with the present experimental found only two binding sites for IHP per bSH conditions. Experiments in which the ab- tetramer. The reason for this dis- sorption of protons by psSH upon IHP bind- crepancy is not clear. It is possible that ing was measured at pH 7.35 and 0.15 M the higher binding constant calculated by Cl- are in support of the low association HETEROTROPIC INTERACTIONS IN /31SH MONOMERS 203 TABLE II CALCULATED VALUES OF KapptD THE APPARENT MONOMER-TETRAMER ASSOCIATION CONSTANT FOR DEOXYCENATED AND OXYGENATED flSH CHAINS AT VARIOUS CONCENTRATIONS OF IHP WPI (mM) 0 1 14 30 60 e? (I/M)3 KFp (1/M)3 1.6 f 0.3 X 1Ol3 7.4 f 1.2 x 1Ol4 1.2 f 0.4 x lOI 8.3 k 2.8 X 1Ol4 4.1 * 0.9 x 10” 6.6 f 1.5 x lOI 1.1 f 0.3 x 10” 1.5 f 0.5 x 10’3 1.1 f 0.1 x 10” 1.5 + 0.2 x 1ol3 a Km Wa,, = i lP~(IHP),I~W%l + I&WP)I}‘) for oxygenated 0 SH chains were calculated as follows: log K”;; = log Kdgy + 4 (log Prmer -log Pgramer). constants reported in this paper (unpub- lished observation). The relatively weak effect of IHP on the monomer-tetramer association constant of flSH chains as shown in Table II is in agreement with the results of Valdes and Ackers (4) who reported that at pH 7.4 IHP, up to a concentration of 1 mM, did not significantly change the monomer-tetramer association constant of deoxy PSH chains. From the fact that the influence of IHP and CO2 on the oxygen affinity of PSH chains is not additional (Fig. 3) it can be concluded that IHP and CO2 compete for a common binding site. Previous results have shown that the effect of CO2 on the oxygen affinity of fldSH requires free N-ter- minal a-amino groups (3). It is likely therefore that in fldSH the binding site for IHP involves the N-terminal a-amino groups which might also be true for fllSH. Last, we wish to discuss the influence of IHP and Pi on the stability of PdSH. From the fact that IHP binds about three times stronger to PISH than to DISH, it can be con- cluded that the presence of IHP favors the dissociation of PdSH into 4 PISH. Thus, IHP destabilizes the BASH homotetramer. From the data given in Table I it can be seen that the oxygen affinity of PSH chains in the presence of 0.05 M Cl- and 0.1 M Pi is equal to that of the undissociated fidSH homotetramer down to a heme concentra- tion of 4 X 10e5 M. At lower protein con- centrations the oxygen affinity of the PSH chains decreases. So it appears that the substitution of 0.1 M Cl- by Pi causes a strong stabilization of fidSH. One explana- tion for this stabilizing action of Pi could be that Pi has a very small affinity for /3iSH and is therefore bound essentially to 84 SH. In this respect it would differ from IHP which was shown to interact effec- tively with the monomeric DISH chains in an oxygen-linked fashion. It is known that allosteric cofactors which reduce the oxygen affinity of human adult hemoglobin (cu&J can alter the ter- tiary structure of the protein (13). The experiments presented in this paper un- equivocally demonstrate the occurrence of heterotropic interactions in isolated mon- omers of the /3 chains. This result is the more remarkable as neither isolated (Y chains nor sperm whale myoglobin exhib- ited such heterotropic interactions. It fol- lows from these results that the tertiary structure of pSH monomers changes upon oxygen binding to such an extent that the affinity for IHP and CO2 significantly de- creases. Therefore, the /3 chains retain some of their basic functional properties which they have in the intact heterotre- tramer (a&) even in the monomeric form. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. REFERENCES BONAVENTURA, J., BONAVENTURA, C., AMICONI, G., TENTORI, L., BRUNORI, M., AND ANTONINI, E. (19’75) J. Biol. Chem. 250, 6278-6281. ROLLEMA, H. S., DE BRUIN, S. H., AND VAN OS, G. A. J. (1976) FEBS Lett. 61.148-150. BAUER, C., AND’KURTZ, A. (1977) J. Biol. Chem. 252,2952-2955. VALDES, R., AND ACKERS, G. K. (1978) Proc. Nat. Acad Sci. USA 75,311-314. KURTZ, A., AND BAUER, C. (1978) B&hem. Bio- phys. Res. Commun. 84.852-857. BAUER, C., AND PACYNA, B. (1975) AnaL Biochem. 65.445-448. BENESCH, R., MACDUFF, G., AND BENESCH, R. E. (1965) Anal. Biochem. 11, 81-87. BAUER, C., BAUMANN, R., ENGELS, U., AND PA- CYNA, B. (1975) J. BioZ. Chem. 250,2173-2176. JELKMANN, W., AND BAUER, C. (1978) PJlfi~ers Arch. 377,75-80. BALDWIN, J. (1975) Progr. Biophys. MoL Biol. 29, 225-320. SZABO, A., AND KARPLUS, M. (1976) Biochemistry 15,2869-2877. SALAHUDDIN, A., AND BUCCI, E. (1976) Biochem- istry 15, 33994405. PERUTZ, M. F. (1976) Brit. Med. Bull. 32,195-208. 

Heterotropic interactions in monomeric beta SH chains from human hemoglobin

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Heterotropic interactions in monomeric beta SH chains from human hemoglobin

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