Proc. Nati. Acad. Sci. USA Vol. 73, No. 12, pp. 4271-4273, December 1976 Chemistry Volume changes in binding of ligands to methemoglobin and metmyoglobin (pressure effects on ligand binding/dilatometry) GABRIEL B. OGUNMOLA*, WALTER KAUZMANNtf, AND ADAM ZIPPt * Department of Chemistry, University of Ibadan, Ibadan, Nigeria; and t Department of Chemistry, Princeton University, Princeton, New Jersey 08540 Contributed by Walter Kauzmann, September 17, 1976

ABSTRACT The volume changes for the binding of various although quite concentrated solutions are required (approxi- ligands to metmyoglobin and methemoglobin have been de- mately 20 g/100 cm3). On the other hand, hemoglobin is ad- termined from the effect of pressure on the binding constants low pressures, undergoing a (for metmyoglobin) and by direct dilatometry (for methemo- versely affected by relatively globin). The volume changes associated with the binding of transition between 1 atm (0.1 MPa) and 2000 atm (200 MPa) cyanide and azide ions to methemoglobin are pH-dependent. (7), which is just the pressure range required for the determi- The volume change for the binding reaction is evidently af- nation of the slope of the plot of In KL against pressure. fected by the same subtle structural variations that have been Metmyoglobin, on the other hand, is much more stable to judged to be present from the variation with pH of enthalpy and pressure, being quite unaffected by pressures well above 2000 entropy for the binding reactions in these proteins. Hydration atm at 6-8 and 2° (8). Therefore, it is possible to determine changes and spin state changes which have been postulated to pH be linked with structural variations in these proteins must be AV for ligand binding to metmyoglobin through the study of pH-dependent. the effect of pressure on the binding constant. The volume changes accompanying the binding of oxygen The volume changes, AV, that result from chemical reactions and ethylisocyanate by ferrohemoglobin have been studied by involving proteins are believed to be influenced by changes in Johnson and Schlegel (9) and by Suzuki et al. (10) from the conformation and hydration that accompany these reactions, effect of hydrostatic pressure on the binding constants. It was but little is known about the relationship between these volume found that AV = 0 for the binding of oxygen and AV = -23 changes and the structural changes in the protein and in the cm3/mol of ligand bound for ethylisocyanate. No studies of the solvent that are responsible for them. effect of pH on AV have as yet been reported, however. The thermodynamics of the binding reactions of different methemoglobins and of sperm whale metmyoglobin with MATERIALS AND METHODS various ligands has been investigated by studying the effect of temperature on the equilibrium constant, KL, for the reaction Human hemoglobin was prepared by the usual method and in which a ligand replaces the water molecule in the sixth oxidized to methemoglobin with 2-fold excess of potassium coordination position of the iron atom in the heme (1-4). The ferricyanide (1). Ferrocyanide and excess ferricyanide were point of interest of this reaction is that although the ligand removed by dialysis against 0.05 M Bis-Tris buffer at pH 6.0. binding reactions of all methemoglobins studied so far show a Concentrated hemoglobin solutions for the dilatometric mea- relatively small dependence of log KL (and therefore of AG) surements were prepared by extended dialysis under pressure on the pH between pH 6 and 9, AH and AS are strongly pH- against appropriate buffer solutions, so that the dialysate was dependent. It has been suggested that these large compensating in osmotic equilibrium with the electrolyte in the protein so- effects might arise in at least two ways: (a) through confor- lution. mational changes in the protein, which would give rise to en- Sperm whale myoglobin, obtained from Sigma, was used thalpy and entropy changes of the same sign, and would without further purification. Buffers used in the pressure ex- therefore compensate at least to some extent (2, 3, 5); and (b) periment were cacodylate and Tris; the Tris buffer was pre- hydration changes, which might give rise to exact compensation pared from Trizma base (Sigma) and Trizma-HCI (Sigma), if water in a hydration sheath underwent a continuous "phase which had been dried under reduced pressure prior to use. change" while always in equilibrium with the solvent water (6). Bis-Tris and Tris buffers were used in the dilatometric exper- Since large volume changes might be expected to accompany iments. either of these two mechanisms, it is of interest to determine All methemoglobin and metmyoglobin solutions studied were whether there is a large pH dependence of AV for the binding 0.05 M in buffer. All measurements were made at 25.0°. reactions of the heme proteins. High Pressure Studies. The high pressure optical bomb was Volume changes of chemical reactions can be most conve- designed by W. B. Daniels. This bomb and other features of the niently measured in two ways for systems that are of inter- high pressure experiment have been described elsewhere (8, est here: by mixing the heme protein and the ligand in a 11, 12). dilatometer, and by the effect of pressure on the binding The ionization constant, K5, of metmyoglobin was deter- constant through the thermodynamic relationship AV = mined spectrophotometrically (1) at pressures of 1, 500, 1000, -RT d In KL/dP. Typically, dilatometry requires rather large 1500, 2000, 2500, and 3000 kg/cm2; spectra were recorded at amounts of reactants (0.1 meq, or of the order of 1 g of heme each pressure for seven different solutions at pH values of 6.2, protein per measurement) so it is not suitable for use with ex- 8.2, 8.4, 8.6, 8.8, 9.0, and 12.0. Corrections to the absorbance pensive proteins such as myoglobin. Fortunately hemoglobin due to compression of the solvent were made using the P-V data is available in sufficient amounts for this not to be a problem, on water of Grindley and Lind (13). The volume change on ionization was found by plotting In KI against the pressure; t To whom requests for reprints should be sent. within the experimental error this plot was a straight line. 4271 Downloaded by guest on September 29, 2021 4272 Chemistry: Ogunmola et al. Proc. Natl. Acad. Sci. USA 73 (1976) The binding constants of azide, fluoride, formate, and im- Table 1. Volume changes for binding reactions of idazole to metmyoglobin were determined by the method de- myoglobin (Mb) from the pressure dependence of the scribed by Anusiem et al. (2). For each ligand, spectra were binding constant recorded at 1, 500, 1000, 1500, and 2000 kg/cm2, at each of 10 concentrations of the ligand. In all measurements above 1 atm, AV, corrections to the absorbance due to compression of the solvent Binding reaction pH ml/mol ligand were made as described above. Values of the binding constant, MbOH, + N- MbN3 + H20 6.0 -8.95 ± 1.8 ,KL, were calculated for each solution at each pressure. For each 7.0 -9.6 ± 3.5 solution, In KL was plotted against the pressure. The mean slope MbOH2 + F-- MbF + H20 6.0 -3.3 ± 1.0 of the straight lines drawn through each set of points was used MbOH2 + formate - Mb- to calculate AV. The error limits reported for AV are estimated formate + H20 6.0 7.5 ± 1.0 from the maximum and minimum slopes consistent with the MbOH2 + imidazole - Mb- data as a whole for each ligand. imidazole + H20 6.0 0.0 ± 2.0 Dilatometric Measurements. Volume changes were found MbOH2 + OH- - MbOH + when 5 ml of buffered methemoglobin solution were mixed in H20 8-9 11.0 ± 1.2* Carlsberg dilatometers with 1 ml of excess ligand in buffer of MbOH2 - MbOH- + H+ 8-9 -11.7 ± 1.2 the same composition as that which was in dialysis equilibrium with the methemoglobin solution during its preparatikn. The * This value was found by adding 23 ml (= AVfor the reaction H20 - H+ + OH-) to the volume change for MbOH2 - MbOH- + dilatometric techniques were those described by Linder- H+. strom-Lang and Lanz (14). For each methemoglobin solution, control experiments were run in which the methemoglobin solutions were mixed with buffer not containing the ligand. The DISCUSSION small volume changes that were observed here are presumably Variation of A V with pH. In interpreting the pH depen- caused by dilution of the concentrated methemoglobin solutions dence of the volume change on binding azide to hemoglobin as well as by readjustments of the ionization of the protein in the pH range 6-8, we must recognize that we are concerned caused by slight pH differences between the protein solution with the two reactions and the buffer. The volume changes observed in these control experiments (generally contractions of a few hundredths of a HbOH2 + L 1- HbL + H20 AV= AV1 [1] microliter) were subtracted from the volume changes obtained HbOH + L + HB+ on adding the ligand at the same pH. The result is the corrected HBL + B + H.,O AV = AV2 [2] volume change, Av. The volume changes, AV, in ml/mol of ligand bound, were calculated from the expression AV = where L is the ligand and HB+ is the acid form of the Bis-Tris 340 Av/[Hbl, where [Hb] is the methemoglobin concentration buffer, B. In the case of the binding of cyanide, since HCN has before mixing in g/100 ml of solution and Av is in microliters. (The number of moles of heme iron present in the dilatometer / is 0.05 [Hb]/17,000.) The methemoglobin concentrations were 20 determined spectrophotometrically as cyanmethemoglobin, using E540 = 10.9 mM-1 cm-1; the rnethemoglobin concen- trations were generally around 20 g/100 ml. We estimate that 8 I *-1-I the values of Av could be reproduced to ±0.03 /d, which leads to an uncertainty of the order of 0.6 ml/mol in our dilatometric AV values. 0~~~~~~~~~~~~~~~0 RESULTS Table 1 gives volume changes for the binding of various ligands E to metmyoglobin, as determined from the effect of pressure on the binding constants and the ionization constant. The precision 0~~~~~~~~~~ of measurement of AV by this method was not sufficient to establish any dependence of AV on the pH in the case of azide binding. Fig. 1 shows the pH dependence of the volume changes for the binding of azide and cyanide to methemoglobin, as deter- mined by dilatometry. It is evident that there is a small pH 42-3 dependence of AV for both ligands between pH 6 and 7.7. In addition, dilatometric measurements were made of the volume changes for the binding of fluoride and formate to methemo- globin. The AV values found were -0.2 ml/mol for formate at pH 6.5 and 4.7 ml/mol for fluoride at pH 6.3. Within the precision of the measurements, the volume changes for the binding of azide by metmyoglobin and methemoglobin are nearly the same (-11 ml/mol). The volume 6.0 6.5 7.0 7.5 changes for the binding of fluoride and formate appear to differ pH by somewhat more than the experimental error for methemo- FIG. 1. pH dependence of the volume changes on binding cyanide globin and metmyoglobin (4.7 and -3.3 ml/mol for fluoride, (0) and azide (-) to human methemoglobin A at 25°. Dashed lines and -0.2 and 7.5 m/mol for formate at pH -6). give volume changes calculated as explained in the text. Downloaded by guest on September 29, 2021 Chemistry: Ogunmola et al. Proc. Natl. Acad. Sci. USA 73 (1976) 4273 a pK = 9.21 at 250 (15), the correspondirig reactions are: states in the heme iron, whereas water, fluoride, and formate HbOH2 + HCN + B produce high-spin states, our results suggest that the conversion of high-spin heme into low-spin heme is accompanied b an HbCN + H20 + BH+ AVAV= AV'1V[1'],Woverall contraction of the protein of something like 10 ml/heme. HbOH + HCN , HbCN + H20 AV = AV'2 [2'] This is equivalent to the removal of a cavity 1.6 A in radius. If we consider, in addition, the reactions This volume change is much larger than that associated with the simple movement of the ferric ion into the heme plane HbOH2 -. HbOH + H+ AV = AV3 [3] when it changes from the high spin to the low spin state. In the HB+ - H+ + B A V = AV4 [4] high spin state the ion is 0.3 A out of the heme plane and its radius is 0.12 A greater than that of the low spin ion. The vol- then we see that ume change associated with the 0.12 A decrease in radius and AV2 = AV1 + iV4 i6 V3 the 0.3 A movement into the 1 A diameter hole in the center of AV'2 = AV'l + AV4 - AV, the heme plane is only of the order of 0.2-0.3 ml/heme. The observed volume chanve clearlv reflects, changes on the nrotein Furthermore, the pK of reaction [3] is 8.1. If AV1, AV2, AV1, molecule other than those in the heme group. AV2, and AV3 are independent of pH, then we should expect that a plot of the volume for the binding of azide or cyanide W.K. received support from the National Science Foundation SEED against the pH would show a sigmoid shape, with AV = AV1 Program in order to work at the University of Ibadan, Nigeria. The (or AV) at low pH and AV = AV2 (or AVV) at high pH and research was also supported by an National Science Foundation Grant with the midpoint of the sigmoid curve at pH 8.1. It is known to Princeton University. G.B.O. was a Fulbright-Hays Visiting Fellow, Princeton University, July-August 1974. W.K. was a Visiting Professor, that AV4 = 1 ml (16). If we assume that AV3 has the same value University of Ibadan, Ibadan, Nigeria, January-March 1975. for hemoglobin as we have measured for myoglobin (-11.7 ml), then AV2 and AV1 would differ by -11 ml (= 1 - 11.7). Curves 1. Beetlestone, J. G. & Irvine, D. H. (1964) Proc. R. Soc. London Ser. showing the variation in AV with pH when these assumptions A 277,401-413. are made are included as dashed lines in Fig. 1 (AV1 has been 2. Anusiem, A. C. I., Beetlestone, J. G. & Irvine, D. H. (1966) J. taken as -11.5 ml and AVI as -17 ml). Chem. Soc. A, 357-363. It is clear that the observed pH dependence of AV for azide 3. Anusiem, A. C. I., Beetlestone, J. G. & Irvine, D. H. (1968) J. and cyanide cannot be explained in this way. One or more of Chem. Soc. A, 960-969. 4. the assumptions that we have made above must therefore be Bailey, J. E., Beetlestone, J. G., Irvine, D. H. & Ogunmola, G. B. (1970) J. Chem. Soc. A, 749-756. incorrect; we are forced to conclude that one or both of the 5. Lumry, R. & Rajender, S. (1970) Biopolymers 9, 1125-1227. following statements is true: (a) either AV1 or AV2, or both, are 6. Ives, D. J. & Marsden, P. S. (1965) J. Chem. Soc., 649-676. rather strongly dependent on the pH (and the same is true of 7. Ogunmola, G. B., Kauzmann, W. & Zipp, A. (1977) Proc. Natl. AV' and AV2), or (b) AV3 must be very different (perhaps of Acad. Sci. USA 74, in press. the order of +10 ml rather than -11.7 ml) for hemoglobin than 8. Zipp, A. & Kauzmann, W. (1972) Biochemistry 12, 4217- it is for myoglobin. Whatever the explanation, it is evident that 4228. AV for these binding reactions must be strongly affected by the 9. Johnson, F. H. & Schlegel, F. M. (1948) J. Cell. Comp. Physiol. same subtle structural variations in these proteins that have been 31,421-425. judged to be present from the pH variation in AH and AS for 10. Suzuki, K., Taniguchi, Y. & Izui, K. (1972) J. Biochem. (Tokyo) the binding reactions (3, 17). 71, 901-904. 11. Kliman, H. L. (1969) Ph.D. dissertation, Princeton University. Relative Values of A V for Different Ligands. From dila- 12. Zipp, A. P. (1973) Ph.D. dissertation, Princeton University. tometric data on mixing aqueous solutions of NaCN and HGI, 13. Grindley, T. & Lind, J. E. (1971) J. Chem. Phys. 54, 3983- we find that AV = -6.5 for the reaction 3989. HCN H+ + CN- 14. Linderstrom-Lang, K. & Lanz, H. (1938) C. R. Trav. Lab. Carlsberg Ser. Chim. 21, 315-338. This means that AV = -9.5 for the reaction 15. Izatt, T. M., Christensen, J. J., Park, R. T. & Bench, R. (1962) HbOH2 + L- HbL + H20 Inorg. Chem. 1, 828-831. 16. Neuman, R. C., Kauzmann, W. & Zipp, A. (1973) J. Phys. Chem. when L- is CN-. This is to be compared with the values -11.5 77,2687-2691. when L- is azide, -0.2 ml when L- is formate, and +4.7 ml 17. Anusiem, A. C. I. & Lumry, R. (1973) J. Am. Chem. Soc. 95, when L- is fluoride. Since azide and cyanide produce low spin 904-908. Downloaded by guest on September 29, 2021