BIOPOLYMERS
VOL. 16, 2613-2618 (1977)
Binding of Azide Ion to Methemoglobin at Elevated Temperatures and the Reality of the "Compensation" Temperature A. C. I. ANUSIEM, G. B. OGUNMOLA, and J. G. BEETLESTONE, Department of Chemistry, University of Ibadan, Ibadan, Nigeria Synopsis We have studied the binding of azide ion to ferrihemoglobinat elevated temperatures. Up to a temperature of 45OC there is no difference in the ligand binding behavior of hemoglobin when compared with the results obtained a t lower temperatures. The compensation temperature Tc of 290.6 f 5.3OK, obtained in this study within the temperature range 30%318'K, confirms that the compensation pattern obtained by Lumry and Rajender is not dependent on the temperature range of the experiment but an intrinsic property of the protein conformation.
The binding of ligand ions to ferrihemoglobin has been extensively studied by Beetlestone and coworkers.14 Some of the major conclusions of these studies are: (1) that each ferrihemoglobin species has a "characteristic pH"-a pH where AH" for the ligand ion binding is a maximum, and (2) a plot of AH" against AS" for the binding that is always linear. Most of these studies were, however, confined to temperatures between 5 and 27°C. Lumry and Rajendar5 later reviewed a number of reactions in which small ions are bound to both small and large molecules, including the binding of small ligand ions to ferrihemoglobin. They showed that for ferrihemoglobin reactions, as well as other binding processes in aqueous medium, a plot of AH" against AS" always generated a straight line with a slope of about 280 f 15"K, which they called the compensation temperature Tc. A possible criticism of the compensation temperature obtained in the case of binding of small ions to ferrihemoglobin is that the experimental temperatures were never far away from Tc values and hence the contributions to AH" and TASO from the compensation process nearly cancel out a t all the experimental temperatures. It therefore became necessary to extend the binding studies to temperatures more removed from the compensation temperature to see if the same compensation pattern is still obtained. We have extended such studies to 45°C and compared these to the results obtained a t lower temperatures. Our results show that even at these elevated temperatures we still obtain a compensation temperature of 290.6 f 5.3"K, in agreement with results obtained at lower temperatures. 2613 0 1977 by John Wiley & Sons, Inc.
2614
ANUSIEM, OGUNMOLA, AND BEETLESTONE
EXPERIMENTAL Materials The preparation of ferrihemoglobin from outdated blood-bank human blood was carried out using the method described by Beetlestone and coworkers.* Phosphate buffer was used in the region below pH 8 and borate buffers above pH 8. The ionic strength of the solutions was adjusted to 0.05 with NaC1. Purified NaN3 obtained from Fischer Scientific Co. was used without further purification.
Determination of Ionization Constant The ionization constant of the water at the sixth coordination position in ferrihemoglobin was determined at various temperatures by the method described by Beetlestone and I r ~ i n eusing ~ . ~ a Unicam SP 500 spectrophotometer with a constant-temperature cell compartment.
Measurement of Equilibrium Constants The equilibrium constant for the binding of azide ion to ferrihemoglobin was determined using the method described by Beetlestone and Irvine.2 To guard against any possible denaturation, equilibration of mixtures containing azide ion and ferrihemoglobin was limited to 2 hr and pH between 5.6 and 8.6. With these limits our results showed that there was attainment of equilibrium and that the Hill constant n was approximately equal to 1.
RESULTS AND DISCUSSION Insofar as can be judged from the spectra of ferrihemoglobin samples within the limits of our experimental conditions, it can be concluded that at elevated temperatures the state of the protein does not change. We may therefore use the same arguments at the elevated temperature as were used
TABLE I Values of pK$ at Various Temperatures ( I = 0.05) t
("0 10 20 30 37 40
45
P d 8.28 f 0.02 8.18 f 0.03 7.99 f 0.01 7.90 f 0.02 7.86 f 0.04 7.78 f 0.02
AZIDE ION BINDING TO METHEMOGLOBIN
2615
at lower temperatures to calculate the equilibrium constants. Table I shows the value of pK3 which is the ionization constant of the water attached to the sixth coordination position of ferrihemoglobin molecule. The values obtained for the different temperatures in this study are slightly different from those reported by Beetlestone and Irvine.6 As mentioned el~ewhere,l*~ the binding of azide ion to ferrihemoglobin is in competition with the ionization of ferrihemoglobin to its alkaline form and thus the two equilibria may be represented by the reaction scheme H+
KCl K + HbOH + Hb+OH2 + N3- +HbN3
(1)
where Hb+OH2, HbOH, and HbN3 represent ferrihemoglobin, alkaline ferrihemoglobin,and ferrihemoglobin azide, respectively. We can therefore define the following equilibrium constants [HbOH][H+] K, = [Hb+OH2] [HbNd =[[HB+OH2] [HbOH]][N3-]
+
It is readily shown that
K = Kobs(l + K,/H+) TABLE I1 Values of Log K at Various Temperatures and pH ( I = 0.05)
t = 30.2OC PH
log K
PH
5.61 5.86 6.02 6.50 6.83 7.23 7.69 7.89
5.26 f 0.03 5.36 f 0.01 5.33 f 0.09 5.32 f 0.05 5.28 f 0.01 5.18 f 0.02 5.11 f 0.04 5.07 f 0.06
5.63 5.86 6.02 6.50 6.84 7.25 7.87 8.31
t = 37.OoC log K 5.18 f 0.01 5.18 f 0.01 5.14 f 0.03 5.06 f 0.02 5.05 f 0.03 4.96 f 0.06 4.78 f 0.05 4.85 f 0.01
t = 45OC PH
log K
5.66 5.84 6.03 6.50 6.88 7.27 7.70 7.85 8.28
5.02 f 0.01 5.02 f 0.02 4.79 f 0.08 4.90 f 0.07 4.82 f 0.04 4.72 f 0.01 4.65 f 0.01 4.64 f 0.01 4.68 f 0.01
TABLE I11 Values of log K Recalculated from Data Given by Beetlestone and Coworkers Using New pK3 Obtained in This Study
t = 2O0C
t = 11.4"C pH
log K
PH
log K
5.42 5.91 6.33 6.65 7.02 7.29 7.88 8.34
5.75 f 0.02 5.77 f 0.04 5.84 f 0.01 5.88 f 0.01 5.83 f 0.01 5.74 f 0.01 5.56 f 0.02 5.33 f 0.01
5.46 5.75 6.30 6.60 6.96 7.27 7.85 8.29
5.52 f 0.02 5.52 f 0.02 5.52 f 0.02 5.52 f 0.01 5.47 f 0.01 5.44 f 0.02 5.37 f 0.01 5.32 f 0.03
2616
ANUSIEM, OGUNMOLA, AND BEETLESTONE
The final value of K used is corrected for hydrazoic acid, which is present at more acid pH values. Tables I1 and I11 show log K at the various temperatures and the recalculated values of log K at the lower temperatures previously obtained' using the new values of pKL obtained in this study. Values of AH"were obtained from the plots of interpolated values of log K a t constant pH against 1/T. Figure 1is a plot of AH" as a function of pH. The same characteristic bell-shaped curve is obtained and the "characteristic pH" (-pH for maximum -AHo) is at pH 7. In Fig. 2 we have plotted -AH"against -ASo and all the points lie on a straight line with slope equal to 290.6 f 5.3"K. It must be pointed out that the values of -AH" obtained in this study are generally lower than those given in an earlier spectrophotometric study,l but agree more closely with the calorimetric data of Anusiem and Lumry.8 This however may be a consequence
Fig. 1. The plot of variation of AHowith pH for human methemoglobin A azide reaction. The AHowas obtained in the temperature range 37-45OC. 16-
1L -
;121
I
10
15 -AHO
20
25
cal I m k ldeg
Fig. 2. Compensation plot of AH' vs A S o for the methemoglobin A azide reaction a t different pH values in the temperature range of 37-45OC.
AZIDE ION BINDING TO METHEMOGLOBIN
2617
of the new data obtained for the pKi in this study. This results in a change in the enthalpy value of +3.4 kcal/mol given by Beetlestone and Irvine6 to a value of about +6 kcal/mol for the ionization of the water at the sixth coordination position. It can be seen that the linear relationship between enthalpy and entropy may be a consequence of a weak temperature-dependence of the heatcapacity change. Our results, coupled with the calorimetric data of Anusiem and Lumry,8 tend to rule out this possibility in the case of ferrihemoglobin. In a more recent study, Krug et aL9Johave analyzed most data reported in literature and have suggested that the observed compensation temperatures may be solely a reflection of the propagation of experimental errors and not chemical effects. However, they set a criterion that which they called a null hypothesis. p is the compensation temperature and T h m is the harmonic mean of the experimental temperatures. They showed that unless this hypothesis could be rejected then the compensation temperature could be regarded as a statistical accident. We have analyzed our thermodynamic data by the method suggested by Krug et al.9J0 and for the AH vs A S plot we obtain a compensation temperature of 290.6 f 5.3"K7 whereas the harmonic mean of our experimental temperatures is 310.3"K. The 95% confidence interval for p for our data set is (302.4, 278.8). We cannot unequivocally assert that on the basis of the Krug analysis this signifies chemical causation, but it does suggest that the compensation observed for ferrihemoglobin reactions is not an experimental artifact and is likely to be of general significance. It is imperative that we look more closely at the protein structure and the effect of liquid water on protein processes to find the source of compensation. Lumry and Rajendar5 have highlighted the significance of the compensation temperature and in fact suggested that there may be a link between the source of compensation and the general chemistry of the system in a phenomenological way. It is also possible that some free energy is provided to the chemical system from the water or water-conformation system. An understanding of the origin of compensation temperature Tc will be important in the understanding of several biological processes in aqueous medium.
References Anusiem, A. C., Beetlestone, J. G. & Irvine, D. H. (1966) J . Chem. SOC.A , 10G112. Beetlestone, J. G. & Irvine, D. H. (1968) J. Chem. SOC.A , 951-959. Anusiem, A. C., Beetlestone, J. G. & Irvine, D. H. (1968) J . Chem. SOC.A , 960-969. Beetlestone, J. G. & Irvine, D. H. (1969) J. Chem. SOC.A , 735-742. 5. Lumry, R. & Rajendar, S. (1970) Biopolymers 9,1125-1227. 6. Beetlestone, J. G. & Irvine, D. H. (1964) Proc. R. SOC.,Ser. A 277,401413. 7 . Beetlestone, J. G. & Irvine, D. H. (1965) J. Chem. SOC.A, 3271-3275.
1. 2. 3. 4.
2618
ANUSIEM, OGUNMOLA, AND BEETLESTONE
8. Anusiem, A. C. I. & Lumry, R. (1973) J. Am. Chem. SOC.95,904-908. 9. Krug, R. R., Hunter, W. G. & Greiger, R. A. (1976) J. Phys. Chem. 80,2335-2340. 10. Krug, R. R., Hunter, W. G. & Greiger, R. A. (1976) J. Phys. Chem. 80,2341-2351.
Received November 29,1976 Accepted April 15,1977
VOL. 16, 2613-2618 (1977)
Binding of Azide Ion to Methemoglobin at Elevated Temperatures and the Reality of the "Compensation" Temperature A. C. I. ANUSIEM, G. B. OGUNMOLA, and J. G. BEETLESTONE, Department of Chemistry, University of Ibadan, Ibadan, Nigeria Synopsis We have studied the binding of azide ion to ferrihemoglobinat elevated temperatures. Up to a temperature of 45OC there is no difference in the ligand binding behavior of hemoglobin when compared with the results obtained a t lower temperatures. The compensation temperature Tc of 290.6 f 5.3OK, obtained in this study within the temperature range 30%318'K, confirms that the compensation pattern obtained by Lumry and Rajender is not dependent on the temperature range of the experiment but an intrinsic property of the protein conformation.
The binding of ligand ions to ferrihemoglobin has been extensively studied by Beetlestone and coworkers.14 Some of the major conclusions of these studies are: (1) that each ferrihemoglobin species has a "characteristic pH"-a pH where AH" for the ligand ion binding is a maximum, and (2) a plot of AH" against AS" for the binding that is always linear. Most of these studies were, however, confined to temperatures between 5 and 27°C. Lumry and Rajendar5 later reviewed a number of reactions in which small ions are bound to both small and large molecules, including the binding of small ligand ions to ferrihemoglobin. They showed that for ferrihemoglobin reactions, as well as other binding processes in aqueous medium, a plot of AH" against AS" always generated a straight line with a slope of about 280 f 15"K, which they called the compensation temperature Tc. A possible criticism of the compensation temperature obtained in the case of binding of small ions to ferrihemoglobin is that the experimental temperatures were never far away from Tc values and hence the contributions to AH" and TASO from the compensation process nearly cancel out a t all the experimental temperatures. It therefore became necessary to extend the binding studies to temperatures more removed from the compensation temperature to see if the same compensation pattern is still obtained. We have extended such studies to 45°C and compared these to the results obtained a t lower temperatures. Our results show that even at these elevated temperatures we still obtain a compensation temperature of 290.6 f 5.3"K, in agreement with results obtained at lower temperatures. 2613 0 1977 by John Wiley & Sons, Inc.
2614
ANUSIEM, OGUNMOLA, AND BEETLESTONE
EXPERIMENTAL Materials The preparation of ferrihemoglobin from outdated blood-bank human blood was carried out using the method described by Beetlestone and coworkers.* Phosphate buffer was used in the region below pH 8 and borate buffers above pH 8. The ionic strength of the solutions was adjusted to 0.05 with NaC1. Purified NaN3 obtained from Fischer Scientific Co. was used without further purification.
Determination of Ionization Constant The ionization constant of the water at the sixth coordination position in ferrihemoglobin was determined at various temperatures by the method described by Beetlestone and I r ~ i n eusing ~ . ~ a Unicam SP 500 spectrophotometer with a constant-temperature cell compartment.
Measurement of Equilibrium Constants The equilibrium constant for the binding of azide ion to ferrihemoglobin was determined using the method described by Beetlestone and Irvine.2 To guard against any possible denaturation, equilibration of mixtures containing azide ion and ferrihemoglobin was limited to 2 hr and pH between 5.6 and 8.6. With these limits our results showed that there was attainment of equilibrium and that the Hill constant n was approximately equal to 1.
RESULTS AND DISCUSSION Insofar as can be judged from the spectra of ferrihemoglobin samples within the limits of our experimental conditions, it can be concluded that at elevated temperatures the state of the protein does not change. We may therefore use the same arguments at the elevated temperature as were used
TABLE I Values of pK$ at Various Temperatures ( I = 0.05) t
("0 10 20 30 37 40
45
P d 8.28 f 0.02 8.18 f 0.03 7.99 f 0.01 7.90 f 0.02 7.86 f 0.04 7.78 f 0.02
AZIDE ION BINDING TO METHEMOGLOBIN
2615
at lower temperatures to calculate the equilibrium constants. Table I shows the value of pK3 which is the ionization constant of the water attached to the sixth coordination position of ferrihemoglobin molecule. The values obtained for the different temperatures in this study are slightly different from those reported by Beetlestone and Irvine.6 As mentioned el~ewhere,l*~ the binding of azide ion to ferrihemoglobin is in competition with the ionization of ferrihemoglobin to its alkaline form and thus the two equilibria may be represented by the reaction scheme H+
KCl K + HbOH + Hb+OH2 + N3- +HbN3
(1)
where Hb+OH2, HbOH, and HbN3 represent ferrihemoglobin, alkaline ferrihemoglobin,and ferrihemoglobin azide, respectively. We can therefore define the following equilibrium constants [HbOH][H+] K, = [Hb+OH2] [HbNd =[[HB+OH2] [HbOH]][N3-]
+
It is readily shown that
K = Kobs(l + K,/H+) TABLE I1 Values of Log K at Various Temperatures and pH ( I = 0.05)
t = 30.2OC PH
log K
PH
5.61 5.86 6.02 6.50 6.83 7.23 7.69 7.89
5.26 f 0.03 5.36 f 0.01 5.33 f 0.09 5.32 f 0.05 5.28 f 0.01 5.18 f 0.02 5.11 f 0.04 5.07 f 0.06
5.63 5.86 6.02 6.50 6.84 7.25 7.87 8.31
t = 37.OoC log K 5.18 f 0.01 5.18 f 0.01 5.14 f 0.03 5.06 f 0.02 5.05 f 0.03 4.96 f 0.06 4.78 f 0.05 4.85 f 0.01
t = 45OC PH
log K
5.66 5.84 6.03 6.50 6.88 7.27 7.70 7.85 8.28
5.02 f 0.01 5.02 f 0.02 4.79 f 0.08 4.90 f 0.07 4.82 f 0.04 4.72 f 0.01 4.65 f 0.01 4.64 f 0.01 4.68 f 0.01
TABLE I11 Values of log K Recalculated from Data Given by Beetlestone and Coworkers Using New pK3 Obtained in This Study
t = 2O0C
t = 11.4"C pH
log K
PH
log K
5.42 5.91 6.33 6.65 7.02 7.29 7.88 8.34
5.75 f 0.02 5.77 f 0.04 5.84 f 0.01 5.88 f 0.01 5.83 f 0.01 5.74 f 0.01 5.56 f 0.02 5.33 f 0.01
5.46 5.75 6.30 6.60 6.96 7.27 7.85 8.29
5.52 f 0.02 5.52 f 0.02 5.52 f 0.02 5.52 f 0.01 5.47 f 0.01 5.44 f 0.02 5.37 f 0.01 5.32 f 0.03
2616
ANUSIEM, OGUNMOLA, AND BEETLESTONE
The final value of K used is corrected for hydrazoic acid, which is present at more acid pH values. Tables I1 and I11 show log K at the various temperatures and the recalculated values of log K at the lower temperatures previously obtained' using the new values of pKL obtained in this study. Values of AH"were obtained from the plots of interpolated values of log K a t constant pH against 1/T. Figure 1is a plot of AH" as a function of pH. The same characteristic bell-shaped curve is obtained and the "characteristic pH" (-pH for maximum -AHo) is at pH 7. In Fig. 2 we have plotted -AH"against -ASo and all the points lie on a straight line with slope equal to 290.6 f 5.3"K. It must be pointed out that the values of -AH" obtained in this study are generally lower than those given in an earlier spectrophotometric study,l but agree more closely with the calorimetric data of Anusiem and Lumry.8 This however may be a consequence
Fig. 1. The plot of variation of AHowith pH for human methemoglobin A azide reaction. The AHowas obtained in the temperature range 37-45OC. 16-
1L -
;121
I
10
15 -AHO
20
25
cal I m k ldeg
Fig. 2. Compensation plot of AH' vs A S o for the methemoglobin A azide reaction a t different pH values in the temperature range of 37-45OC.
AZIDE ION BINDING TO METHEMOGLOBIN
2617
of the new data obtained for the pKi in this study. This results in a change in the enthalpy value of +3.4 kcal/mol given by Beetlestone and Irvine6 to a value of about +6 kcal/mol for the ionization of the water at the sixth coordination position. It can be seen that the linear relationship between enthalpy and entropy may be a consequence of a weak temperature-dependence of the heatcapacity change. Our results, coupled with the calorimetric data of Anusiem and Lumry,8 tend to rule out this possibility in the case of ferrihemoglobin. In a more recent study, Krug et aL9Johave analyzed most data reported in literature and have suggested that the observed compensation temperatures may be solely a reflection of the propagation of experimental errors and not chemical effects. However, they set a criterion that which they called a null hypothesis. p is the compensation temperature and T h m is the harmonic mean of the experimental temperatures. They showed that unless this hypothesis could be rejected then the compensation temperature could be regarded as a statistical accident. We have analyzed our thermodynamic data by the method suggested by Krug et al.9J0 and for the AH vs A S plot we obtain a compensation temperature of 290.6 f 5.3"K7 whereas the harmonic mean of our experimental temperatures is 310.3"K. The 95% confidence interval for p for our data set is (302.4, 278.8). We cannot unequivocally assert that on the basis of the Krug analysis this signifies chemical causation, but it does suggest that the compensation observed for ferrihemoglobin reactions is not an experimental artifact and is likely to be of general significance. It is imperative that we look more closely at the protein structure and the effect of liquid water on protein processes to find the source of compensation. Lumry and Rajendar5 have highlighted the significance of the compensation temperature and in fact suggested that there may be a link between the source of compensation and the general chemistry of the system in a phenomenological way. It is also possible that some free energy is provided to the chemical system from the water or water-conformation system. An understanding of the origin of compensation temperature Tc will be important in the understanding of several biological processes in aqueous medium.
References Anusiem, A. C., Beetlestone, J. G. & Irvine, D. H. (1966) J . Chem. SOC.A , 10G112. Beetlestone, J. G. & Irvine, D. H. (1968) J. Chem. SOC.A , 951-959. Anusiem, A. C., Beetlestone, J. G. & Irvine, D. H. (1968) J . Chem. SOC.A , 960-969. Beetlestone, J. G. & Irvine, D. H. (1969) J. Chem. SOC.A , 735-742. 5. Lumry, R. & Rajendar, S. (1970) Biopolymers 9,1125-1227. 6. Beetlestone, J. G. & Irvine, D. H. (1964) Proc. R. SOC.,Ser. A 277,401413. 7 . Beetlestone, J. G. & Irvine, D. H. (1965) J. Chem. SOC.A, 3271-3275.
1. 2. 3. 4.
2618
ANUSIEM, OGUNMOLA, AND BEETLESTONE
8. Anusiem, A. C. I. & Lumry, R. (1973) J. Am. Chem. SOC.95,904-908. 9. Krug, R. R., Hunter, W. G. & Greiger, R. A. (1976) J. Phys. Chem. 80,2335-2340. 10. Krug, R. R., Hunter, W. G. & Greiger, R. A. (1976) J. Phys. Chem. 80,2341-2351.
Received November 29,1976 Accepted April 15,1977
