The kinetics of formation of the asymmetric carbon-monoxyhemoglobin hybrid (\u3b1\u3b2)(A)(\u3b1\u3b2)(C) from the parent molecules \u3b12\u3b22(A) and \u3b12\u3b22(C) have been studied by electrophoresis at subzero temperatures (down to -40\ub0C) using as supporting media gels of acrylamide/methylacrylate in dimethyl sulfoxide/water mixtures. It has been found that in these media the rate of hybrid formation is markedly affected by pH and decreases by an order of magnitude between pH 7.3 and 8.3. At pH>10, t=-40\ub0C, the hybrid between \u3b12\u3b22(A) and \u3b12\u3b22(C) is stable for several hours. A rapid thermal quenching of a mixture of \u3b12\u3b22(A) and \u3b12\u3b22(C) prevented hybrid formation during the time required to separate the 2 molecules

L. Rossi Bernardi

M. Perrella

M. Samaja

The kinetics of formation of the asymmetric carbon-monoxyhemoglobin hybrid (αβ)(A)(αβ)(C) from the parent molecules α2β2(A) and α2β2(C) have been studied by electrophoresis at subzero temperatures (down to -40°C) using as supporting media gels of acrylamide/methylacrylate in dimethyl sulfoxide/water mixtures. It has been found that in these media the rate of hybrid formation is markedly affected by pH and decreases by an order of magnitude between pH 7.3 and 8.3. At pH>10, t=-40°C, the hybrid between α2β2(A) and α2β2(C) is stable for several hours. A rapid thermal quenching of a mixture of α2β2(A) and α2β2(C) prevented hybrid formation during the time required to separate the 2 molecules

AIR Universita degli studi di Milano

Communication Hybrid Formation for Liganded Hemoglobins A and C at Subzero Temperatures* (Received for publication, April 5, 1979, and in revised form, June 22, 1979) Michele Perrella, Michele Samaja, and Luigi Rossi- Bernardi From the Catted-a di Enzimologia, University of Milan, Via G. Celoria, 2, 20133 Milnn, Italy The kinetics of formation of the asymmetric carbon- monoxyhemoglobin hybrid (~~)A(c$)C from the parent molecules (Y&S* and a#zC have been studied by electro- phoresis at subzero temperatures (down to -40°C) us- ing as supporting media gels of acrylamide/methyla- crylate in dimethyl sulfoxide/water mixtures. It has been found that in these media the rate of hybrid for- mation is markedly affected by pH and decreases by an order of magnitude between pH 7.3 and 8.3. At pH >lO, t = -4O”C, the hybrid between a&” and (Y&~ is stable for several hours. A rapid thermal quenching of a mix- ture of azPzA and w&” prevented hybrid formation during the time required to separate the 2 molecules. Isoelectric focusing in polyacrylamide gels has been exten- sively used (l-3) to study a variety of asymmetrical hemoglo- bin hybrids, which are formed when two different hemoglobins of unlike charge are mixed together. Since the isoelectric focusing technique is intrinsically slow, all previous studies on this problem have been confined mainly to unliganded he- moglobin, whose dimerization rate is orders of magnitude slower than that of liganded hemoglobin. We have recently reported (4) that electrophoretic or isoe- lectric focusing separations can be achieved at subzero tem- peratures (-30 to -40°C) by using as supporting media gels of acrylamide/methylacrylate copolymers and dimethyl sulf- oxide/water mixtures, and we have preliminarily shown that, at low temperatures, hybrid species, which are formed upon mixing human carbonmonoxyhemoglobins A and S, are stable enough to allow their isolation by this technique. More de- tailed studies on this problem may not only shed further light on the dimerization reaction of liganded hemoglobin, but also promise to indicate appropriate experimental conditions for the isolation of intermediates in the reaction of hemoglobin with oxygen or other ligands. In fact, it has been shown (5) that the “off’ reaction of oxygen or carbon monoxide from hemoglobin is considerably slowed down at low temperatures. The stabilization of intermediate compounds of hemoglobin with ligands by a thermal quenching approach may lead to their isolation, if such intermediates do indeed exist in non- vanishing concentration, and if suitable methods of separation at low temperature can be used. Bunn and McDonough (3) and Park (2) have tried to detect * This work was supported by a grant from the Consiglio Nazion& delle Ricerche, Rome. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. intermediate compounds in the reaction of carbon monoxide with hemoglobin by focusing samples of human hemoglobin partially saturated with carbon monoxide or nitric oxide, but they observed only the separation of fully liganded from fully unliganded hemoglobin, which was possible because of the charge difference due to the Bohr effect. The conditions used in their experiments, however, did not exclude the possibility of ligand exchange reactions between tetramers during the separation process, either by direct ligand reaction exchange or, indirectly, by the dimerization and reassociation reactions of fully or partly liganded hemoglobin. In this paper, we report studies on the effect of temperature on the rate of dimerization of human carbonmonoxyhemoglo- bin. Detailed information on this reaction is, indeed, a prereq- uisite to any attempt to isolate intermediates in the reaction of hemoglobin with ligands, such as carbon monoxide or oxygen, by physical separation methods. EXPERIMENTAL PROCEDURES Hemoglobin A,) and hemoglobin C were obtained from blood sam- ples from a normal and an A-C heterozygote donor and were purified by ion exchange chromatography (6). The hemoglobin used in this work, unless otherwise stated, has been stored and used in the carbonmonoxy form. Electrophoresis at -4O’C was carried out as described by Perrella et al. (4). The eel-containing tubes were refrig- erated by the circulation of a coolant thermostatted at -43°C. The eel temnerature during the senaration was -40 + 1°C. Gels were preparei by copolyme&ation of acrylamide and methylacrylate in a 1:l molar ratio, ?“W, = 8.44, and methylenebisacrylamide as the cross- linking agent, C’% = 1.82 (4). Gel and electrodic buffers contained 50% (v/v) MerSO.’ The gel buffer was 43 mM Tris and 7.6 mM HCl, and the electrodic buffer was 68.5 mM glycine and 8.6 mM Tris. Their respective pH values at -40°C were approximately 10.5 f 0.3 and 10.3 f 0.3. These pH values were obtained by the method of the dye indicators described by Douzou (7). The reported values are approximate because they were extrapolated from pH data measured at 0, -10, -20, and -30°C. It should be added that these buffers are 4 to 7 times more concentrated than those studied by Douzou. Samples of HbAo and HbC of identical concentrations (about 0.4 mM in heme) were incubated as follows. Each sample of HbAo or HbC was prepared in aqueous 20 mM phosphate or Tris buffer (7). Solid sucrose was added to each hemoglobin solution to a final 10% (w/v) concentration to increase the viscosity and density of the solution. The samples of HbAo or HbC were then introduced into two separate wells at the bottom of a thermostatted elass vessel. When the tem- perature of the two samples reached Oat, an equal volume of cold (OOC) ethylene glycol was-added, and the temperature of the solutions was then brought to the reauired value. Mixing was obtained by manually shaking the vessel for 1 or 2 min. Incubation times were considered to start upon shaking the vessel and terminated upon deliverv of the samales into the seoaration cell at -4O’C. Sample delivery was carried out by the use of a microsyringe tipped with a Teflon capillary tube of about lo-p1 capacity, which was precooled at the temuerature of senaration. Twenty to twenty-five _ micrograms of protein were loaded on each gel tube. Electrophoresis was carried out for 2 to 2% h at a constant piwer of about 0.05 watt/ gel and 850 V. At the end of the seaaration. the gel-containing tubes were removed from the cell and microphotographed. Scans of pho- tographic prints were obtained with a Zeiss MQ3 densitometer. Areas under the peaks were integrated by planimetry. Pictures in which the I The abbreviations used are: MesSO, dimethyl sulfoxide; EGOH, ethylene glycol; HbAo, a&“, hemoglobin & CO form. HBC (Y /3 ‘~ hemoglobin C, CO form; HbS, hemoglobin 4, CO for;; (cY&($~~: the hybrid hemoglobin species resulting from the dimerization and recombination of a dimer from Hb& and a dimer from HbC. 8748  at Università degli studi di Milano, on January 11, 2010www.jbc.orgDownloaded from Hemoglobin Hybrids at Subzero Temperatures 8749 ratio of peak areas corresponding to HbAo and HbC was found to exceed the range 1 + 0.1 were discarded, since the two hemoglobins were used in equimolar amounts. RESULTS AND DISCUSSION The amino acid substitution present in HbC (@o’” * Lv*) makes the mixture of HbAo and HbC used in this work well suited for the electrophoretic separation of the hybrid species from the parent molecules, since it allows a larger charge difference among the three species than that possible with other systems, such as a mixture of HbG, and hemoglobin S (4). Since HbAo and HbC have practically identical oxygen binding and other functional properties and the amino acid substitution involves a residue located on the surface of the molecule, it can be assumed, in accordance with Bunn and McDonough (3), that the rate constant for the dimerization reaction is similar, if not the same, for the two parent mole- cules and the hybrid molecule, and that HbAo, HbC, and their hybrid species have identical thermodynamic stability. It has been shown (3) that, following the above assumptions, the fist order constant for the hemoglobin tetramer dissocia- tion, h, can be calculated from the slope of a plot of -ln(l - ~YH) uersus time, where YH is the fraction of hybrid species present in an equimolar mixture of HbAo and HbC at the incubation time t. Fig. la is a photograph of a gel showing the electrophoretic separation at -40°C of an equimolar mixture of HbAo and HbC, both in the carbonmonoxy form. The mixture was incubated for 15 min at 0°C in 10 mM phosphate, 50% (v/v) EGOH, pH 7.2. Preliminary experiments have shown that, during this incubation period, the formation of the hybrid species, (@)*(cyp)c, practically goes to equilibrium. The ex- perimental conditions used for the electrophoretic separation of the three species, a$3zA, (Y&‘, and (tip)*(&)“, e.g. T = -4O”C, pH > 10, time approximately 2 h, are likely to prac- tically stop dimer exchange between the three species. This is proved by the fact that the middle band, ($?)*(~yp)~, corre- sponds to half (+4%) the total hemoglobin, a finding which is a b C FIG. 1. Electrophoretic separation. a, electrophoretic separa- tion of an equimolar mixture of carbonmonoxy HbAo and HbC, incubated at 0°C for 15 min and then separated at -4O’C. b, same as a, but after separation in three bands the sample was left in the gel for another 2 h, followed by an additional 2-h period of separation. c, expected, on statistical grounds, if complete equilibrium be- tween the three species had been reached during the incuba- tion time and if no appreciable dissociation of the hybrid species had occurred during the electrophoretic separation at -40°C. This situation is confirmed by the electrophoretic separations reported in Fig. 1, b and c. In the experiment reported in Fig. lb, electrophoresis of a sample of HbAo and HbC was carried out under the same experimental conditions as described for Fig. la. After sepa- ration in three bands, the sample was left in the gel for another 2 h, followed by an additional 2-h period of separation. No decrease in the relative proportion of the middle band was found, indicating that during the equilibration period of 2 h in the gel at -40°C no appreciable formation of a&” and C&Z’ from (ap)“(a/3)” has occurred. The experiment reported in Fig. lc confirms the conclusion reached above. In this case, two samples of HbAo and HbC, 10 mM in phosphate, 50% (v/v) EGOH, pH 7.5, were mixed (about 2 min) at -30°C in the thermostatted glass vessel and then separated by electrophoresis at -4O”C, pH > 10, in about 2 h. No hybrid species is apparent. This lack of a middle hybrid species was found even if the mixture was left on top of the gel for 1 h. Only increasing the equilibration time at -40°C to 2 h caused the appearance of a very faint middle band. It should be noted that the time required for the sample to enter the gel, and thus to change its pH from about 7.5 to over 10, is, at -40°C about 30 to 45 min. Fig. 2 shows logarithmic plots of (1 - ZYH) for samples containing equimolar concentrations of HbAo and HbC mixed at -10°C and left for various incubation times (abscissa). Fig. 2a refers to a sample in 10 mM phosphate, 50% (v/v) EGOH, pH 7.3, whereas Fig. 2b refers to identical conditions except for the pH of the incubating mixture, which was = 8.3. Similar t (min) FIG. 2. Logarithmic plots of (1 - 2 YH) for samples containing equimolar concentrations of HbAo and HbC, mixed at -10°C electrophoretic separation of two samples of HbAo and HbC, mixed at -3O’C and then separated at -40°C in about 2 h. and left for various incubation times. a, pH 7.3; b, pH 8.3.  at Università degli studi di Milano, on January 11, 2010www.jbc.orgDownloaded from 8750 Hemoglobin Hybrids at Subzero Temperatures TABLE I Values of the apparent velocity constant, k, for the formation of the hybrid (~@)*(a/?)” from carbonmonoxy a&~~ and a&” PH T  k (miK’) X 10” “C 7.3 -5 95 7.3 -10 55 7.35 -14 23 7.4 -21 14 7.45 -26 11 8.3 -10 4.7 9.6” -10 1.4 significantly affected by the presence of EGOH and by the variations in the dielectric constant at various subzero tem- ” 10 nm Tris-HCl buffer, 50’S (v/v) EGOW. All other data were obtained using 10 nm phosphate buffers, 50% (v/v) EGOH. plots, obtained for experiments in which the temperature of incubation and the pH was varied, allowed the calculation of k, the apparent first order rate constant, for the rate of dissociation of tetramers into dimers (Table I). The apparent activation energy, E,, for the dimerization reaction in 10 mM phosphate buffer, pH 7.3,50% (v/v) EGOH, as calculated from the data of Table I, neglecting the slight pH change of the phosphate buffer over the temperature range considered, is about 13.5 kcal/mol. A marked decrease in the rate constant with increasing pH is observed at subzero temperatures (Fig. 2 and Table I). This effect is in qualitative agreement with the experiments reported by Flamig and Parkhurst (8) for aqueous carboxyhemoglobin at 215°C. Pre- sumably, the interpretation of the effect of pH on the decrease in the rate of dimerization given by these authors, e.g. for- mation of new coulombic interactions between charged groups at alkaline pH, also applies under the experimental conditions reported in this paper. The reader is cautioned against assigning to the values of k, as obtained in this paper, a true physicochemical meaning. In fact, it should be noted that the dielectric constant of 50% (v/ v) EGOH in water varies with temperature. Since the forces governing the rate of tetramer dissociation are at least in part electrostatic, it is likely that the absolute value of k will be peratures. On the other hand, the values of k reported in Table I and the corresponding apparent energy of activation for the di- merization reaction under the solvent conditions used repre- sent useful empirical information for planning experiments for the separation of hybrid species between two different li- ganded hemoglobins (Fig. la) or for attempting the demon- stration of intermediate compounds of hemoglobin with lig- ands by this technique. It is relevant to point out that, during the time (about 2 h) required for separation at -40°C (Fig. 1, a, b, and c), no hybrid resulting from the dissociation-associ- ation reaction of the two parent tetramers was found. It should be noted that the pH value of the hemoglobin inside the gel is about 10. At this rather alkaline pH and subzero tempera- ture (-4O”C), it can safely be assumed that no significant association-dissociation reaction between two different he- moglobin species or between intermediate species (e.g. Hb4XI, Hb4X2, Hb4X3, or Hb4X4) is likely to occur during the time required to carry out an electrophoretic separation by the technique used. REFERENCES 1. Bunn, H. F. (1972) in Hemoglobin and Red Cell Structure and Function (Brewer, G., ed) pp. 41-53, Plenum Press, New York 2. Park, C. M. (1973) Ann. N. Y. Acad. Sci. 209,237-256 3. Bunn, H. F., and McDonough, M. (1974) Biochemistry 13, 988- 993 4. Perrella, M., Heyda, A., Mosca, A., and Rossi-Bernardi, L. (1978) Anal. Biochem. 88,212-224 5. Douzou. P. (1977) Adv. Enzvmol. Relat. Areas Mol. Biol. 45. 250-254 6. Huinsman, T. H. J., and Dozy, A. M. (1965) J. Chromatogr. 19, 160-169 7. Douzou, P. (1977) Cryobiochemistry, pp. 49-75, Academic Press, New York 8. Flamig, D. P., and Parkhurst, L. T. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 3814-3816  at Università degli studi di Milano, on January 11, 2010www.jbc.orgDownloaded from 

Hybrid formation for liganded hemoglobins A and C at subzero temperatures

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Hybrid formation for liganded hemoglobins A and C at subzero temperatures

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