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Differential scanning calorimetry of the thermal denaturation of lactate dehydrogenase
Bioehimiea et Biophysica Acta, 493 (1977) 142-153
© Elsevier/North-Holland Biomedical Press BBA 37701 D I F F E R E N T I A L S C A N N I N G C A L O R I M E T R Y OF T H E T H E R M A L DENAT U R A T I O N OF LACTATE D E H Y D R O G E N A S E
A. L. JACOBSON and H. BRAUN Biochemistry Group, Department of Chemistry, The University of Calgary, Calgary, Alberta, T2N 1N4 (Canada)
(Received December 20th, 1976)
SUMMARY 1. Differential scanning calorimetry has been used to study the thermal denaturation of lactate dehydrogenase. At pH 7.0 in 0.1 M potassium phosphate buffer, only one transition was observed. Both the enthalpy of denaturation and the melting temperature are linear function of heating rate. The enthalpy is 430 kcal/mol and the melting temperature 61 °C at 0 °C/min heating rate. The ratio of the calorimetric heat to the effective enthalpy indicated that the denaturation is highly cooperative. Subunit association does not appear to significantly contribute to the enthalpy of denaturation. 2. Both cofactor and sucrose addition stabilized the protein against thermal denaturation. Pyruvate addition produced no changes. Only a small time-dependent destabilization was observed at low concentrations of urea. Large effects were observed in concentrated NaC1 solutions and with sulfhydryl-modified lactate dehydrogenase.
INTRODUCTION Differential scanning calorimetry is a very sensitive method of measuring enthalpy differences between a sample and a reference material. Calorimetric studies of denaturation can provide additional information concerning the mechanisms of denaturation in proteins. Lumry et al. [1] and Tanford [2] have shown that the cooperativity of the transition in thermal denaturation can be determined by comparison of the calorimetric heat and the van't Hoff transition enthalpy. A number of workers have determined the van't Hoff transition enthalpy by assuming a two-state transition occurs, and that the area under the thermogram at a particular temperature is proportional to the amount denatured [3-8]. Privalov and coworkers [9-13] use the heat capacity at the transition temperature to determine an effective enthalpy. The comparison of the calorimetric heat to the effective enthalpy is then used to determine the "cooperativity" or nature of the transition. For a number of globular proteins, the cooperativity indicates that a simple two-state model can be used to describe the thermal denaturation. The proteins for which a two-state denaturation model can be used include lysozyme [3-6, 9, 10, 12], ribonuclease [4, 7, 9, 10, 12], myoglobin [9, 12]; chymotrypsinogen [5, 9, 13, 14], and cytochrome c [12].
143 There have been a number of reports of proteins in which the thermal denaturation is complex. Donovan and Mikalyi [15] reported two endothermal transitions in fibrinogen which indicates that portions of the structure undergo thermal denaturation independently. Privalov et al. [13] have shown that the melting curves for tRNA T M are complex and that there are six essentially independent cooperative regions in this structure. With several other specific transfer RNAs, the thermal transitions are also not cooperative enough to be described by a two-state model [8]. There are relatively few calorimetric studies of the denaturation of oligomeric proteins. The enthalpy changes reported for avidin [16] are larger than for the simpler protein systems (in terms of kcal/mol). Cofactor binding drastically increases the thermal stability of avidin. There was no indication of dissociation of subunits in the thermal denaturation of avidin. Donovan and Beardslee [17] found that the melting temperature increased for the trypsin.soybean trypsin inhibitor complex compare to the individual proteins, but that the enthalpy of denaturation was essentially additive. Lactate dehydrogenase is a well characterized complex enzyme [18], and hence particularly well suited for thermal studies. Cho and Swaisgood [19] have shown that there is a slow concentration-dependent dissociation of the tetrameric enzyme to dimer with the rabbit muscle enzyme. The dissociation is slight above 4-5 mg/ml, and pronounced at less than 1 mg/ml. Cofactor binding stabilizes the enzyme. There are four equivalent non-interacting binding sites for NADH/tetramer unit [20]. The concentration-dependent dissociation of subunits [19] as well as the dissociation by sodium dodecyl sulfate [21] are inhibited by cofactor binding. X-ray analysis [18, 22] shows that binding of cofactor causes a conformational change within the protein. Hinz and Jaenicke [23] have shown that the enthalpy of association of NADI-I with pig skeletal muscle lactate dehydrogenase is exothermic with a large negative temperature coefficient. Urea cleaves lactate dehydrogenase into subunits only at high concentration [24] exposing additional sulfhydryl groups [25]. Sulfhydryl groups are situated in each subunit near the active site [18] and the reactivity of sulfhydryl groups appears to correlate to spontaneous dissociation of the tetramer at low protein concentration [19] and to loss of enzymatic activity without dissociation of the tetramer at higher protein concentration [25]. In this work the enthalpy associated with thermal denaturation of lactate dehydrogenase, has been studied. The effect ofcofactor, substrate, sulfhydryl reagents, sucrose, salt, and urea on the enthalpy and on the rate of thermal denaturation has been determined. EXPERIMENTAL Rabbit muscle lactate dehydrogenase (crystalline type II) was purchased from Sigma Chemical Co. and dialized against 0.1 M potassium phosphate buffer at pH 7.0. The protein samples were concentrated with an Amicon concentrator. The molecular weight was taken as 141 000. The protein concentration was calculated by assuming the extinction coefficient of a 1 ~ solution was 12.3 at 280 nm [26]. A Perkin-Elmer differential scanning calorimeter Model II was used for the thermal measurements. The aluminum pans supplied by Perkin-Elmer, teflon-coated A 1 pans (prepared by coating the Perkin-Elmer with teflon spray), and Dupont-coated
144 A1 hermetic pans were all tested to determine if the A1 pans affected the protein. No differences in spectra were observed with all types of pans. Samples were weighed into pans (-I- 1 #g) and runs started at 10 °C. The reproducibility of the enthalpy calculations was considerably higher if both sample and reference buffer were both weighed in the pans rather than the pipetted volume (15/~1) used to determine the amount of protein in the pan. The concentrated protein solution was viscous and difficult to pipette reproducibily. With the Perkin-Elmer pans, reproducible enthalpy measurements were obtained at all heating rates. However, these pans were difficult to seal and leaks frequently developed. The runs in which leaks occurred were discarded and the instrument heated and recalibrated after each leaky sample. With the coated pans, more reproducible enthalpy values were obtained when the heating rate was 2.5 °C/ min or lower. The coating appeared to affect the thermal contact between the sample and the cell. However, with these lower heating rates the enthalpy calculated was reproducible. The calorimeter was calibrated using indium, benzoic acid and azobenzene. Areas were measured both with a planimeter and by weight. Peak area was determined by using a straight line under the thermogram connecting the base line of the onset of denaturation to the end of the denaturation. The enthalpy of the denaturation (AH) was calculated from peak area and known standards. The temperature at the peak of the thermogram was considered the melting temperature (Tin). The effective enthalpy was calculated by the method of Privalov and Khechinashvili [12]. The activation energy was calculated by the method suggested by Beech [27]. In this method the rate of heat flow into the sample is assumed proportioned to the change in fraction denatured. Sedimentation data was obtained at a protein concentration of 10 mg/ml (which is lower than the concentration used in the calorimeter) with a Beckman Model E ultracentrifuge. Disc gel electrophoresis was at pH 8.6 in a 5 ~ acrylamide gel. The protein concentration for sulfhydryl modifications and for the electrophoresis was also 10 mg/ml. RESULTS Sample thermograms for lactate dehydrogenase are shown in Fig. 1. At a heating rate of 10 °C/min the transition appears symmetrical and there is no indication of any intermediate transitions. With a heating rate of 2.5 °C/min the transition appears less symmetrical but there is still no indication of any distinct intermediate transitions (or additional transitions at higher temperatures) which might be associated with dissociation of subunits preceding denaturation. At a constant heating rate the thermograms for lactate dehydrogenase are reproducible and both the temperature at the peak of the thermogram (Tm) and the enthalpy of thermal denaturation (AH) are independent of the protein concentration (19-94 mg/ml). The thermal denaturation is not reversible when the protein is heated to 70 °C. After cooling and reheating precipitation occurs. The precipitation is an exothermic process. The precipitation was confirmed by following changes in a glass vial and by opening pans after reheating. Addition of 10 mM dithiothreitol did not affect either the non-reversibility of the denaturation or the precipitation. However, reversible partial denaturation does occur when the protein solution is heated to temperatures
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TEMPERATURE (*K) Fig. 1. Effect of heating rate on the thermograms of lactate dehydrogenase in 0.10 M potassium phosphate buffer at pH 7.0. Top heating rate of 10 °C/min. The sample contained 10 mg of a 42.5 mg/ml of protein. Bottom heating rate of 2.5 °C/min. The sample contained 14.6 mg of a 48.6 mg/ml protein solution. below Tin. This partial denaturation is totally reversible if the protein is heated to several degrees below Tin. The extent of the reversibility depends on how close Tm is approached and the heating and cooling rates. The reversibility is higher at the higher heating rates. Both Tm and A H are linear functions of heating rate (measured at 10, 5, 2.5 °C/min). Extrapolation to 0 heating rate gives a Tm of 61 °C and a A H of 430 kcal/ tool. The melting temperatures and the enthalpies of denaturation of lactate dehydrogenase are summarized in Table I. The protein is stable in concentrated solution at room temperature for 30 min. There is no change in Tm or A H during this time period. There is also essentially no change in either Tm or A H when an excess of substrate, pyruvate, is added to the system. In presence of pyruvate, both Tm and d H are dependent on heating rate in the same manner as was the pure lactate dehydrogenase solution. Sucrose stabilized lactate dehydrogenase from heat denaturation. The melting temperature was increased 8 °C. However, the enthalpy of the transition was not affected by sucrose addition. Cofactor addition also stabilized the protein from thermal denaturation. In this case both the calorimetric enthalpy and the melting temperature are increased by the addition of N A D H . In these experiments the cofactor N A D H was always in excess and the [NADH]/[protein] ratio varied between 22 and 230.
146 TABLE I THERMAL DENATURATION OF LACTATE DEHYDROGENASE AT pH 7.0 IN 0.1 M POTASSIUM PHOSPHATE BUFFER
AH
/IH
z~Heff
(cal/g)
(kcal/mol)
(kcal/mol)
66.7 4- 0.2 62.2 4- 0.2 62.2 4- 0.2
3.6 4- 0.3 3.2 4- 0.3 3.3 ± 0.3
508 ± 42 451 ± 56 465 ± 45
491 4- 28 503 4- 40 460 2- 40
1.0 0.9 1.0
10 2.5 10
66.7 ± 0.2 61.4 2- 0.2 66.7 4- 0.2
3.5 ± 0.2 3.0 ± 0.2 3.5 i 0.2
495 ± 20 428 :L 23 495 2- 20
465 ± 20 499 ± 16 473 2- 24
1.1 1.0 1.0
Sucrose 1 mM
10
74.3 2- 0.2
3.6 2- 0.2
508 ± 28
445 2- 28
1.0
NADH 3 mM 30 mM 100 mM
10 10 10
71.8 2- 0.3 71.9 ± 0.3 70.5 ± 0.5
4.3 ± 0.2 4.2 ± 0.2 4.0 ± 0.2
606 ± 28 592 ± 28 564 ± 31
501 2_ 40 494 ± 40 448 2- 40
1.2 1.2 1.2
10 2.5 2.5~ i0
65.7 54.2 54.7 65.0
3.3 2.9 2.5 3.0
465 405 352 423
427 469 465 429
± 24 ± 30 4- 28 4- 43
1.1 0.9 0.8 1.0
369 2- 15 269 ± 22
0.7 0.6
Additions a
None
Pyruvate 10mM 100 mM
Urea 1M
2M NaC1 2M 4M
Heating rate (°C/min)
T,~ (°C)
l0 b 2.5 c 2.5 d
2.5 2.5
± 0.2 ± 0.4 4- 0.3 2- 0.4
60.2 -- 0.3 55.0 ± 0.4
± 0.2 ± 0.4 4- 0.3 4- 0.2
1.9 4- 0.3 1.2 4- 0.2
± ± ± ±
14 28 28 28
271 2- 40 172 ± 10
z~H/AI"[efr
a All samples were freshly prepared from concentrated stock solution except as noted. The values are from an average of six samples with the exception of the lactate dehydrogenase standards. b Average of 17 samples. c Average of 22 samples. d Incubated 30 min at 25 °C before calorimetric measurements. Incubated 30 min at 2 °C before calorimetric measurements.
A H e f f w a s calculated at Tm by the m e t h o d o f Privalov and K h e c h i n a s h v i l i [12] and the values are also shown in T a b l e I. T h e ratio o f AH/AHeff (calculated f r o m the average o f the individual runs) was 1.0 ± 0.1 for lactate dehydrogenase and for the samples containing pyruvate a n d sucrose at a heating rate o f 10 °C/min. It is possible that the heating rate could affect this ratio. T h e tendency appears to be for a lower value (0.9) at the 2.5 ° C / m i n heating rate b u t this is within the range o f standard deviation for the 10 ° C / m i n heating rate. This ratio was 1.2 ± 0.1 for lactate dehyd ro g en as e in the solutions c o n t a i n i n g N A D H . C o n s i d e r i n g standard deviations, this value is just ~vithin the experimental error limits, b u t the slightly higher values did occur with all three N A D H concentrations. T h e effect o f various chemical denaturants on the t h e r m a l d e n a t u r a t i o n o f lactate dehydrogenase is also s h o w n in Table I. W i t h freshly prepared samples in I M urea, there is a small decrease in Tm (1 °C) and in A H (8 %) at a heating rate o f 10 °C/min. A d d i t i o n o f 2 M urea produces only a small further decrease. Th er e is
147 essentially no change in the ratios of dH/dHeff on addition of 1-2 M urea. At a heating rate of 2.5 °C/min there is an 8 °C decrease in Tm and a l0 ~o change in AH. The effect of urea is dependent on the contact time of the urea with the protein. After a 30 min incubation at 2 °C, a further 13 ~ decrease in AH was observed though no further changes in Tm were apparent. The changes produced by addition of 2 and 4 M NaC1 are also shown in Table I. With 2 M NaC1 there is only a small change on Tm (--2 °C) while the changes in AH (--40 ~ ) and in AHeff (--27 ~ ) are large. With 4 M NaC1 the destabilization is even larger. The ratios of Z~H/AHef f a r e significantly less than one. The Arrhenius plot of the reaction rate calculated by the method of Beech [27] is shown in Fig. 2. The type of agreement obtained from various experiments is also shown in this figure. In contrast to the calorimetric heat, there is no significant difference in the activation energy (172 ± 5 kcal/mol) when the heating rate is increased from 2.5 to l0 °C/min. The Arrhenius plots are linear to Tm. Only at higher temperatures is there any deviation from linearity. This deviation could be due to the
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Fig. 2. Arrhenius plots of the thermal denaturation of lactate dehydrogenase in 0.10 M potassium phosphate buffer at pH 7.0. C is the calibration constant for the calorimeter. The open symbols are from thermograms at 2.5 °C/min. The samples contained 10 mg of a 42.5 mg/ml protein. The filled symbols are from thermograms at 10 °C/rain. These samples contained 10-15 mg of 42-86 mg/ml protein solution.
148 irreversible denaturation and to inaccuracies in measurement of the small areas at high degrees of denaturation. There was no significant change in the activation energy on addition of pyruvate, sucrose or N A D H . The activation energy decreased by 10 ~o in the urea solutions. There was a 20 ~ decrease in the activation energy after addition of 2 M NaC1 and a 34 ~ decrease after addition of 4 M NaC1. The time dependence of the changes on the enthalpy of denaturation after reaction with n-ethylmaleimide is shown in the bottom portion of Fig. 3. When 2.2.10 -4 M n-ethylmaleimide is added, the ratio of sulfhydryl reagent to the total in sulfhydryl content of the lactate dehydrogenase is 1/4, and with 4.4.10- + M sulfhydryl reagent this ratio is 1/2. The total number of sulfhydryl groups in lactate dehydrogenase was calculated from the value in 8 M urea. The enthalpy of denaturation decreases after n-ethylmaleimide addition. At least 1 h at 25 °C is necessary for complete reaction. A concomitant decrease is seen in the ratio dH/dHeff. There was no change in the melting temperature after the sulfhydryl reagent addition.
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Fig. 3. Time dependence of the reaction of n-ethylmaleimide with lactate dehydrogenase. The ratio of the calorimetric heat to the effective enthalpy of activation is given in the upper l~ortion of the figure and the effect on the calorimetric heat in the lower part of the figure. ©, 2.2 × 10-4 M n-ethylmaleimide, 51 mg/ml protein, the ratio of sulthydryl reagent to protein sulthydryl is 1/4. n, 4.4 × 10-4 M n-ethylmaleimide, 51 mg/ml protein, the ratio of sulfydryl reagent to protein sulfhydryl is 1/2.
149 A comparison o f the effects produced by modifying lactate dehydrogenase with n-ethylmaleimide and with p-hydroxymercuribenzoate is shown in Fig. 4. In these experiments the protein samples were treated with the sulfhydryl reagents for 3--4 h at 4 °C before the calorimetric measurements. The melting temperature is shown at the top o f Fig. 4. With b o t h sulfhydryl reagents there was no change in the Tm when half the sulfhydryl in the protein were titrated. There was only a very small change in Tm (0.7 °C) at full sulfhydryl titration. The enthalpy changes are shown in the b o t t o m o f Fig. 4. The enthalpy o f denaturation decreased 30-60~o after reaction with n-ethylmaleimide, and 70-85
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Fig. 4. Changes due to modification of the sulfhydryl in lactate dehydrogenase (LDH) by n-ethylmaleimide or p-hydroxymercuribenzoate. The protein was assumed to contain 16 sulfhydryl groups/ mol (from titration in urea). The reaction mixture was incubated 4 h at 0 °C before the thermograms. The reaction mixtures contained 20-54 mg/ml protein and the sulfhydryl reagent concentrations were varied to give the appropriate ratios. Average values from six determinations. For the Tm plot: O, n-ethylmaleimide; [], p-hydroxymercuribenzoate, For the AH/A Heft plot : O, n-ethylmaleimide; II, p-hydroxymercuribenzoate. For the AH, dHerr and Ea plot: n-ethylmaleimide: AH, ©, Ancrr, V, E~,- ........ • p-Hydroxymercuribenzoate: AH, II, AHefr; 0, E.,, -........ .
150 after the p-hydroxymercuribenzoate treatment. AHeff also decreased after sulfhydryl modification of the protein, but the changes were of a lower order of magnitude than the changes in AH. As a consequence of these relative changes, the ratio AH/AHeff (shown in the middle portion of Fig. 5) decreased from a ratio of 1.0 ± 1 to 0.7 ~ 0.1 with n-ethylmaleimide and to 0.4 ~ 0.1 with p-hydroxymercuribenzoate. The changes in the activation energy are also shown in the bottom of Fig. 4. There is no change at 1/4 sulfhydryl titration. At full sulfhydryl titration the activation energy is decreased by 35 ~ and the values are similar to the value for lactate dehydrogenase in 4 M NaC1. The calorimetric experiments were at high protein concentration. (Required because of the small volume of the calorimetric cells). At these high protein concentrations there was no observable dissociation of the lactate dehydrogenase at 25 °C even at full titration of the sulfhydryl groups. The sedimentation measured by ultracentrifugal analysis was identical before and after full sulfhydryl titration. The protein appeared at the top of the 5 ~ acrylamide gels and there were no additional bands after full sulfhydryl titration. The gels were heavily loaded and any monomeric lactate dehydrogenase would have appeared in the middle of the gels. DISCUSSION The enthalpy of thermal denaturation of lactate dehydrogenase is 430 kcal/mol from a linear extrapolation to 0 °C/min heating rate. This value is considerably higher than the values previously reported for simple globular proteins [3, 4, 6, 10-12, 17] and for avidin [16]. This relatively high value for the enthalpy of denaturation of lactate dehydrogenase simply reflects its higher molecular weight. If the enthalpy change is expressed in calories/g, the value for lactate dehydrogenase is lower than the values reported for the simpler globular proteins. The enthalpy of denaturation of lactate dehydrogenase per tool of subunit (107 kcal/mol at 0 °C/min heating rate) is of the same order of magnitude as the value for lysozyme which has approximately the same degree of secondary structure. Hence there does not appear to be any large contribution to the total enthalpy of denaturation of lactate dehydrogenase to the interactions between subunits. It is possible that interactions between subunits in a protein does not in general, produce any large contributions to the total enthalpy of denaturation. Donovan and Beardslee [17] found that the melting temperature increased for the trypsin, soybean trypsin inhibitor complex and the trypsin, ovomucoid complex compare to the individual proteins, but the enthalpy of denaturation was essentially additive. In the denaturation of lactate dehydrogenase, both dissociation into subunits and loss of secondary structure in the monomer units should occur. Only one transition was observed. It is possible that the denatured state in lactate dehydrogenase is the denatured tetramer. Ribonuclease, lysozyme and chymotrypsinogen all have been reported to retain some degree of ordered structure after heat denaturation [28]. An alternative possibility is that dissociation is not a rate-limiting step and that the observed heat includes both loss of secondary structure and dissociation of subunits. As in the ease of the enthalpy of thermal denaturation, the activation energy of the lactate dehydrogenase denaturation is considerably higher (172 kcal/mol) than values reported for simpler proteins. Crescenzi and Delben [5] used the Beech method for calculating activation energies and obtained values of 90 kcal/mol for ribo-
151 nuclease, 101 kcal/mol for lysozyme and 80 kcal/mol for chymotrypsinogen A. Donovan and coworkers [16, 17] also used the method of Beech and found values of 69 kcal/mol for soybean trypsin inhibitor, 63 kcal/mol for trypsin, and 90 kcal/mol for avidin. The high value for the activation energy for lactate dehydrogenase probably reflects its high molecular weight as the value considered per mol of monomer is lower than the values for the simpler proteins. The small change in activation energy of lactate dehydrogenase in solutions containing urea is similar in magnitude to the changes reported for ribonuclease and lysozyme at low urea concentrations [5]. The very much larger changes in activation energy of lactate dehydrogenase in concentrated NaC1 solutions and for the sulfhydryl-modified protein correspond in magnitude to the change in the values obtained on addition of guanidine. HC1 to ribonuclease or lysozyme [5]. The very small changes in Tm, and E a for the thermal denaturation of freshly prepared protein at 10 °C/min in 1 and 2 M urea are in general agreement with previous reports that urea cleaves lactate dehydrogenase into subunits only at high concentrations [24]. However, the changes observed at the lower heating rate suggest that there is a slow modification by low concentrations of urea, and the modifications are both time and temperature dependent. Sucrose is well known to stabilize proteins. The stabilization of lactate dehydrogenase by sucrose is most noticeable in the increased Tm ( + 7.6 °C) while there is no change in the enthalpy. Sucrose appears to prevent the onset of denaturation while not affecting the mechanism of denaturation. There have been numerous reports of the stabilization of lactate dehydrogenase by cofactor binding [18]. Hence, stabilization against thermal denaturation was expected and observed. Since the protein melts at a higher temperature, some increase in A H due to the higher temperature is expected. However, the increase in the Tm in the presence of NADH (5 °C) is less than the increase in the presence of sucrose (8 °C). Within experimental error (4-42 kcal/mol) there is no increase in A H in the presence of sucrose. Hence the effect on A H of the increased Tm in the NADH solutions is expected to be small and the degree of stabilization of lactate dehydrogenase against thermal denaturation by its cofactor NADH appears to be directly related to the enthalpy of binding of the cofactor. From an extrapolation of the calorimetric data of Hinz and Jaenicke [23] from 40 °C, the value for AHbt,ding of N ADH to lactate dehydrogenase would be of order of magnitude --90 to --120 kcal/ mol at 71 °C. A lower estimation can also be made from the kinetic data of Borgmann et al. [29]. However, the interpolation from this data is more difficult since equations for variation of ZlHblndlng with temperature were not given. An extrapolation of the calorimetric data for NAD binding to glyceraldehyde-3-phosphate dehydrogenase [30] to 71 °C would give a value of --140 kcal/mol for four NAD bound. This suggests the binding of the cofactor, either N A D H or NAD, is strongly exothermic and of comparable order of magnitude with proteins requiring this cofactor. With lactate dehydrogenase, the increase of 85 kcal/mol in AH in the presence of NADH can easily be accounted for by the dissociation of bound NADH from the protein. The situation is quite different from the binding of biotin to avidin [16] where stabilization is very much greater than expected from the heats of binding of biotin to avidin, and change in heat capacity were postulated to explain the very high degree of stabilization. The stabilization of lactate dehydrogenase to thermal denaturation in the
152 presence of its cofactor N A D H , and the absence of any stabilization by the substrate pyruvate could be due to binding of substrate without any concomitant conformational change in the protein as occurs with cofactor binding, or to very weak binding of substrate. However, these data do suggest that binding of substrate may occur only after binding of cofactor. This latter suggestion has been made previously as part of the proposed mechanism o f enzymatic action [31, 32]. The interpretation of the thermal behaviour of lactate dehydrogenase after modification of sulfhydryl groups appears to be complex. The melting temperature after sulfhydryl modification is not decreased as is the case after urea or NaCI addition to the enzyme solution. Titration of sulfhydryl groups might have been expected to affect the dissociation of subunits and hence the Tin, since the dissociation and reassociation of tetramer at low protein concentrations is dependent on the concentration of free sulfhydryl groups and protective agents such as dithiothreitol increase the reassociation [19]. The disc gel electrophoresis and the ultracentrifugal data clearly show that the initial state is still tetrameric. It is not known whether the final thermally denatured states are the same with and without sulfhydryl modifications of the lactate dehydrogenase. Some changes in enthalpy are expected because of modification of the tetrameric initial state. Differences in cooperativity between subunits would also be expected because of the proximity of the sulfhydryl groups to the sites of association between monomers [18, 19]. The changes in Tm suggest that dissociation into subunits may occur in the NaC1 solution, or in the urea solution on standing, but that such dissociation is not a major factor in the sulfhydryl-modified protein. The higher magnitude of the change in d H with p-hydroxymercuribenzoate to n-ethylmaleimide modification could be due to differences in accessibilities of the protein binding sites to these reagents. However, in both sulfhydryl modifications the enthalpy of thermal denaturation is decreased while Tm change is very small. Privalov and Khechivashvili [12] have shown that if it is assumed that the denaturation represents a two-state transition to which the van't Hoff equation is applied, an effective enthalpy can be calculated at the midpoint of the transition. For a two-state reversible transition this effective enthalpy would be the van't Hoff enthalpy. The ratio of the calorimetric enthalpy to the effective enthalpy would be equal to 1 for a two-state transition, greater than one if there is a significant population of intermediate denatured states and less than one if there are non-equilibrium conditions in the cell due to irreversible denaturation. Since the total denaturation of lactate dehydrogenase is irreversible, values of AH/AHe, less than one are predicted. Values less than one are found in the presence of high concentrations of NaC1 and on standing in urea and for the sulfhydryl-modified lactate dehydrogenase. However, for the pure lactate dehydrogenase (and with the pyruvate and sucrose additions) the ratio of dH/dHeff is 1.0 ~ 0.1. In these cases values less than one also should be found since the total denaturation is irreversible when the protein is heated to 70 °C. The observed values of 1.0 ± 0.1 could be fortuitous, and the actual values less than 1.0 within the limits of experimental accuracy. However, this observed value may reflect the reversible nature of the denaturation below Tin. At temperatures above Tin, the reversibility could critically depend on factors such as temperature of heating, time of heating and cooling, and exchange or oxidation of sulfhydryl groups. The precipitation which occurs after heating to 70 °C and cooling and reheating suggests that some aggregation has occurred. The observed value of 1.0 suggests that the two-
153 state d e n a t u r a t i o n m o d e l for lactate dehydrogenase m a y be a p p r o p r i a t e u n d er specified conditions. ACKNOWLEDGEMENT S u p p o r t f r o m grants f r o m the N a t i o n a l R e s e a r c h C o u n c i l o f C a n a d a and the A l b e r t a H e a r t F o u n d a t i o n are gratefully acknowledged. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Lumry, R., Biltonen, R. and Brandts, J. F. (1966) Biopolymers 4, 917-944 Tanford, C. (1968) Adv. Protein Chem. 23, 122-282 Delben, F. and Crescenzi, V. (1969) Biochim. Biophys. Acta 194, 615-619 Delben, F., Crescenzi, V. and Quadrifoglio, F. (1969) Int. J. Protein Res. 1, 145-149 Crescenzi, V. and Delben, F. (1971) Int. J. Protein Res. 3, 57-62 Reilly, J. M. and Karasz, F. E. (1970) Biol:olymers 9, 1429-1435 Tsong, T. Y., Hearn, R. P., Wrathall, D. P. and Sturtevant, J. M. (1970) Biochemistry 9, 26662677 Brandts, J. F., Jackson, W. M. and Yao-Chung Ting, T. (1974) Biochemistry 13, 3595-3600 Privalov, P. L., Khechinashvili, N. N. and Atanasov, B. P. (1971) Biopolymers 10, 1865-1890 Khechinashvili, N. N., Privalov, P. L. and Tiktopulo, E. I. (1973) FEBS Lett. 30, 57-60 Privalov, P. L., Tiktopulo, E. I. and Khechinashvili, N. N. (1973) Int. J. Peptide Protein Res. 5, 229-237 Privalov, P. L. and Khechinashvili, N. N. (1974) J. Mol. Biol. 86, 665-684 Privalov, P. L., Filimonov, V. V., Venkstern, T. V. and Bayev, A. A. (1975) J. Mol. Biol. 97, 279-288 Jackson, W. M. and Brandts, J. F. (1970) Biochemistry 9, 2294-2301 Donovan, J. W. and Mikalyi, E. (1974) Prov. Natl. Acad. Sci. U.S. 71, 4125-4128 Donovan, J. W. and Ross, K. D. (1973) Biochemistry 12, 512-517 Donovan, J. W. and Beardslee, R. A. (1975) J. Biol. Chem. 250, 1966-1971 Everse, J. and Kaplan, N. O. (1973) Adv. Enzymol. 37, 61-133 Cho, I. C. and Swaisgood, H. (1973) Biochemistry 12, 1572-1577 Kolb, D. and Weber, G. (1975) Biochemistry 14, 4471-4476 DiSabato, G. and Kaplan, N. O. (1964) J. Biol. Chem. 239, 438-443 Adams, M., Haas, D. J., Jeffery, B. A., McPherson, Jr., A., Mermall, H. L., Rossmann, M. G., Schevitz, R. W. and Wonocott, A. J. (1969) J. Mol. Biol. 41, 159-188 Hinz, H. J. and Jaenicke, R. (1975) Biochemistry 14, 24-27 Apella, E. and Markert, C. L. (1961) Biochem. Biophys. Res. Commun. 6, 171-176 DiSabato, G. and Kaplan, N. O. (1963) Biochim. Biophys. Acta 77, 135-138 Fromm, J. (1967) J. Biol. Chem. 238, 2938-2944 Beech, G. (1969) J. Chem. Soc. (A) 1903-1905 Aune, K. C., Salahuddin, A., Zarlengo, M. H. and Tanford, C. (1967) J. Biol. Chem. 242, 44864489 Borgmann, U., Laidler, K. J. and Moon, T. W. (1975) Can. J. Biochem. 53, 1196-1206 Velick, S. F., Baggott, J. P. and Sturtevant, J. M. (1971) Biochemistry 10, 779-786 Schwert, G. W., Miller, B. R. and Peanasky, R. J. (1967) J. Biol. Chem. 242, 3245-3252 Griffin, H. J. and Criddle, R. S. (1970) Biochemistry 9, 1195-1205
© Elsevier/North-Holland Biomedical Press BBA 37701 D I F F E R E N T I A L S C A N N I N G C A L O R I M E T R Y OF T H E T H E R M A L DENAT U R A T I O N OF LACTATE D E H Y D R O G E N A S E
A. L. JACOBSON and H. BRAUN Biochemistry Group, Department of Chemistry, The University of Calgary, Calgary, Alberta, T2N 1N4 (Canada)
(Received December 20th, 1976)
SUMMARY 1. Differential scanning calorimetry has been used to study the thermal denaturation of lactate dehydrogenase. At pH 7.0 in 0.1 M potassium phosphate buffer, only one transition was observed. Both the enthalpy of denaturation and the melting temperature are linear function of heating rate. The enthalpy is 430 kcal/mol and the melting temperature 61 °C at 0 °C/min heating rate. The ratio of the calorimetric heat to the effective enthalpy indicated that the denaturation is highly cooperative. Subunit association does not appear to significantly contribute to the enthalpy of denaturation. 2. Both cofactor and sucrose addition stabilized the protein against thermal denaturation. Pyruvate addition produced no changes. Only a small time-dependent destabilization was observed at low concentrations of urea. Large effects were observed in concentrated NaC1 solutions and with sulfhydryl-modified lactate dehydrogenase.
INTRODUCTION Differential scanning calorimetry is a very sensitive method of measuring enthalpy differences between a sample and a reference material. Calorimetric studies of denaturation can provide additional information concerning the mechanisms of denaturation in proteins. Lumry et al. [1] and Tanford [2] have shown that the cooperativity of the transition in thermal denaturation can be determined by comparison of the calorimetric heat and the van't Hoff transition enthalpy. A number of workers have determined the van't Hoff transition enthalpy by assuming a two-state transition occurs, and that the area under the thermogram at a particular temperature is proportional to the amount denatured [3-8]. Privalov and coworkers [9-13] use the heat capacity at the transition temperature to determine an effective enthalpy. The comparison of the calorimetric heat to the effective enthalpy is then used to determine the "cooperativity" or nature of the transition. For a number of globular proteins, the cooperativity indicates that a simple two-state model can be used to describe the thermal denaturation. The proteins for which a two-state denaturation model can be used include lysozyme [3-6, 9, 10, 12], ribonuclease [4, 7, 9, 10, 12], myoglobin [9, 12]; chymotrypsinogen [5, 9, 13, 14], and cytochrome c [12].
143 There have been a number of reports of proteins in which the thermal denaturation is complex. Donovan and Mikalyi [15] reported two endothermal transitions in fibrinogen which indicates that portions of the structure undergo thermal denaturation independently. Privalov et al. [13] have shown that the melting curves for tRNA T M are complex and that there are six essentially independent cooperative regions in this structure. With several other specific transfer RNAs, the thermal transitions are also not cooperative enough to be described by a two-state model [8]. There are relatively few calorimetric studies of the denaturation of oligomeric proteins. The enthalpy changes reported for avidin [16] are larger than for the simpler protein systems (in terms of kcal/mol). Cofactor binding drastically increases the thermal stability of avidin. There was no indication of dissociation of subunits in the thermal denaturation of avidin. Donovan and Beardslee [17] found that the melting temperature increased for the trypsin.soybean trypsin inhibitor complex compare to the individual proteins, but that the enthalpy of denaturation was essentially additive. Lactate dehydrogenase is a well characterized complex enzyme [18], and hence particularly well suited for thermal studies. Cho and Swaisgood [19] have shown that there is a slow concentration-dependent dissociation of the tetrameric enzyme to dimer with the rabbit muscle enzyme. The dissociation is slight above 4-5 mg/ml, and pronounced at less than 1 mg/ml. Cofactor binding stabilizes the enzyme. There are four equivalent non-interacting binding sites for NADH/tetramer unit [20]. The concentration-dependent dissociation of subunits [19] as well as the dissociation by sodium dodecyl sulfate [21] are inhibited by cofactor binding. X-ray analysis [18, 22] shows that binding of cofactor causes a conformational change within the protein. Hinz and Jaenicke [23] have shown that the enthalpy of association of NADI-I with pig skeletal muscle lactate dehydrogenase is exothermic with a large negative temperature coefficient. Urea cleaves lactate dehydrogenase into subunits only at high concentration [24] exposing additional sulfhydryl groups [25]. Sulfhydryl groups are situated in each subunit near the active site [18] and the reactivity of sulfhydryl groups appears to correlate to spontaneous dissociation of the tetramer at low protein concentration [19] and to loss of enzymatic activity without dissociation of the tetramer at higher protein concentration [25]. In this work the enthalpy associated with thermal denaturation of lactate dehydrogenase, has been studied. The effect ofcofactor, substrate, sulfhydryl reagents, sucrose, salt, and urea on the enthalpy and on the rate of thermal denaturation has been determined. EXPERIMENTAL Rabbit muscle lactate dehydrogenase (crystalline type II) was purchased from Sigma Chemical Co. and dialized against 0.1 M potassium phosphate buffer at pH 7.0. The protein samples were concentrated with an Amicon concentrator. The molecular weight was taken as 141 000. The protein concentration was calculated by assuming the extinction coefficient of a 1 ~ solution was 12.3 at 280 nm [26]. A Perkin-Elmer differential scanning calorimeter Model II was used for the thermal measurements. The aluminum pans supplied by Perkin-Elmer, teflon-coated A 1 pans (prepared by coating the Perkin-Elmer with teflon spray), and Dupont-coated
144 A1 hermetic pans were all tested to determine if the A1 pans affected the protein. No differences in spectra were observed with all types of pans. Samples were weighed into pans (-I- 1 #g) and runs started at 10 °C. The reproducibility of the enthalpy calculations was considerably higher if both sample and reference buffer were both weighed in the pans rather than the pipetted volume (15/~1) used to determine the amount of protein in the pan. The concentrated protein solution was viscous and difficult to pipette reproducibily. With the Perkin-Elmer pans, reproducible enthalpy measurements were obtained at all heating rates. However, these pans were difficult to seal and leaks frequently developed. The runs in which leaks occurred were discarded and the instrument heated and recalibrated after each leaky sample. With the coated pans, more reproducible enthalpy values were obtained when the heating rate was 2.5 °C/ min or lower. The coating appeared to affect the thermal contact between the sample and the cell. However, with these lower heating rates the enthalpy calculated was reproducible. The calorimeter was calibrated using indium, benzoic acid and azobenzene. Areas were measured both with a planimeter and by weight. Peak area was determined by using a straight line under the thermogram connecting the base line of the onset of denaturation to the end of the denaturation. The enthalpy of the denaturation (AH) was calculated from peak area and known standards. The temperature at the peak of the thermogram was considered the melting temperature (Tin). The effective enthalpy was calculated by the method of Privalov and Khechinashvili [12]. The activation energy was calculated by the method suggested by Beech [27]. In this method the rate of heat flow into the sample is assumed proportioned to the change in fraction denatured. Sedimentation data was obtained at a protein concentration of 10 mg/ml (which is lower than the concentration used in the calorimeter) with a Beckman Model E ultracentrifuge. Disc gel electrophoresis was at pH 8.6 in a 5 ~ acrylamide gel. The protein concentration for sulfhydryl modifications and for the electrophoresis was also 10 mg/ml. RESULTS Sample thermograms for lactate dehydrogenase are shown in Fig. 1. At a heating rate of 10 °C/min the transition appears symmetrical and there is no indication of any intermediate transitions. With a heating rate of 2.5 °C/min the transition appears less symmetrical but there is still no indication of any distinct intermediate transitions (or additional transitions at higher temperatures) which might be associated with dissociation of subunits preceding denaturation. At a constant heating rate the thermograms for lactate dehydrogenase are reproducible and both the temperature at the peak of the thermogram (Tm) and the enthalpy of thermal denaturation (AH) are independent of the protein concentration (19-94 mg/ml). The thermal denaturation is not reversible when the protein is heated to 70 °C. After cooling and reheating precipitation occurs. The precipitation is an exothermic process. The precipitation was confirmed by following changes in a glass vial and by opening pans after reheating. Addition of 10 mM dithiothreitol did not affect either the non-reversibility of the denaturation or the precipitation. However, reversible partial denaturation does occur when the protein solution is heated to temperatures
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TEMPERATURE (*K) Fig. 1. Effect of heating rate on the thermograms of lactate dehydrogenase in 0.10 M potassium phosphate buffer at pH 7.0. Top heating rate of 10 °C/min. The sample contained 10 mg of a 42.5 mg/ml of protein. Bottom heating rate of 2.5 °C/min. The sample contained 14.6 mg of a 48.6 mg/ml protein solution. below Tin. This partial denaturation is totally reversible if the protein is heated to several degrees below Tin. The extent of the reversibility depends on how close Tm is approached and the heating and cooling rates. The reversibility is higher at the higher heating rates. Both Tm and A H are linear functions of heating rate (measured at 10, 5, 2.5 °C/min). Extrapolation to 0 heating rate gives a Tm of 61 °C and a A H of 430 kcal/ tool. The melting temperatures and the enthalpies of denaturation of lactate dehydrogenase are summarized in Table I. The protein is stable in concentrated solution at room temperature for 30 min. There is no change in Tm or A H during this time period. There is also essentially no change in either Tm or A H when an excess of substrate, pyruvate, is added to the system. In presence of pyruvate, both Tm and d H are dependent on heating rate in the same manner as was the pure lactate dehydrogenase solution. Sucrose stabilized lactate dehydrogenase from heat denaturation. The melting temperature was increased 8 °C. However, the enthalpy of the transition was not affected by sucrose addition. Cofactor addition also stabilized the protein from thermal denaturation. In this case both the calorimetric enthalpy and the melting temperature are increased by the addition of N A D H . In these experiments the cofactor N A D H was always in excess and the [NADH]/[protein] ratio varied between 22 and 230.
146 TABLE I THERMAL DENATURATION OF LACTATE DEHYDROGENASE AT pH 7.0 IN 0.1 M POTASSIUM PHOSPHATE BUFFER
AH
/IH
z~Heff
(cal/g)
(kcal/mol)
(kcal/mol)
66.7 4- 0.2 62.2 4- 0.2 62.2 4- 0.2
3.6 4- 0.3 3.2 4- 0.3 3.3 ± 0.3
508 ± 42 451 ± 56 465 ± 45
491 4- 28 503 4- 40 460 2- 40
1.0 0.9 1.0
10 2.5 10
66.7 ± 0.2 61.4 2- 0.2 66.7 4- 0.2
3.5 ± 0.2 3.0 ± 0.2 3.5 i 0.2
495 ± 20 428 :L 23 495 2- 20
465 ± 20 499 ± 16 473 2- 24
1.1 1.0 1.0
Sucrose 1 mM
10
74.3 2- 0.2
3.6 2- 0.2
508 ± 28
445 2- 28
1.0
NADH 3 mM 30 mM 100 mM
10 10 10
71.8 2- 0.3 71.9 ± 0.3 70.5 ± 0.5
4.3 ± 0.2 4.2 ± 0.2 4.0 ± 0.2
606 ± 28 592 ± 28 564 ± 31
501 2_ 40 494 ± 40 448 2- 40
1.2 1.2 1.2
10 2.5 2.5~ i0
65.7 54.2 54.7 65.0
3.3 2.9 2.5 3.0
465 405 352 423
427 469 465 429
± 24 ± 30 4- 28 4- 43
1.1 0.9 0.8 1.0
369 2- 15 269 ± 22
0.7 0.6
Additions a
None
Pyruvate 10mM 100 mM
Urea 1M
2M NaC1 2M 4M
Heating rate (°C/min)
T,~ (°C)
l0 b 2.5 c 2.5 d
2.5 2.5
± 0.2 ± 0.4 4- 0.3 2- 0.4
60.2 -- 0.3 55.0 ± 0.4
± 0.2 ± 0.4 4- 0.3 4- 0.2
1.9 4- 0.3 1.2 4- 0.2
± ± ± ±
14 28 28 28
271 2- 40 172 ± 10
z~H/AI"[efr
a All samples were freshly prepared from concentrated stock solution except as noted. The values are from an average of six samples with the exception of the lactate dehydrogenase standards. b Average of 17 samples. c Average of 22 samples. d Incubated 30 min at 25 °C before calorimetric measurements. Incubated 30 min at 2 °C before calorimetric measurements.
A H e f f w a s calculated at Tm by the m e t h o d o f Privalov and K h e c h i n a s h v i l i [12] and the values are also shown in T a b l e I. T h e ratio o f AH/AHeff (calculated f r o m the average o f the individual runs) was 1.0 ± 0.1 for lactate dehydrogenase and for the samples containing pyruvate a n d sucrose at a heating rate o f 10 °C/min. It is possible that the heating rate could affect this ratio. T h e tendency appears to be for a lower value (0.9) at the 2.5 ° C / m i n heating rate b u t this is within the range o f standard deviation for the 10 ° C / m i n heating rate. This ratio was 1.2 ± 0.1 for lactate dehyd ro g en as e in the solutions c o n t a i n i n g N A D H . C o n s i d e r i n g standard deviations, this value is just ~vithin the experimental error limits, b u t the slightly higher values did occur with all three N A D H concentrations. T h e effect o f various chemical denaturants on the t h e r m a l d e n a t u r a t i o n o f lactate dehydrogenase is also s h o w n in Table I. W i t h freshly prepared samples in I M urea, there is a small decrease in Tm (1 °C) and in A H (8 %) at a heating rate o f 10 °C/min. A d d i t i o n o f 2 M urea produces only a small further decrease. Th er e is
147 essentially no change in the ratios of dH/dHeff on addition of 1-2 M urea. At a heating rate of 2.5 °C/min there is an 8 °C decrease in Tm and a l0 ~o change in AH. The effect of urea is dependent on the contact time of the urea with the protein. After a 30 min incubation at 2 °C, a further 13 ~ decrease in AH was observed though no further changes in Tm were apparent. The changes produced by addition of 2 and 4 M NaC1 are also shown in Table I. With 2 M NaC1 there is only a small change on Tm (--2 °C) while the changes in AH (--40 ~ ) and in AHeff (--27 ~ ) are large. With 4 M NaC1 the destabilization is even larger. The ratios of Z~H/AHef f a r e significantly less than one. The Arrhenius plot of the reaction rate calculated by the method of Beech [27] is shown in Fig. 2. The type of agreement obtained from various experiments is also shown in this figure. In contrast to the calorimetric heat, there is no significant difference in the activation energy (172 ± 5 kcal/mol) when the heating rate is increased from 2.5 to l0 °C/min. The Arrhenius plots are linear to Tm. Only at higher temperatures is there any deviation from linearity. This deviation could be due to the
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Fig. 2. Arrhenius plots of the thermal denaturation of lactate dehydrogenase in 0.10 M potassium phosphate buffer at pH 7.0. C is the calibration constant for the calorimeter. The open symbols are from thermograms at 2.5 °C/min. The samples contained 10 mg of a 42.5 mg/ml protein. The filled symbols are from thermograms at 10 °C/rain. These samples contained 10-15 mg of 42-86 mg/ml protein solution.
148 irreversible denaturation and to inaccuracies in measurement of the small areas at high degrees of denaturation. There was no significant change in the activation energy on addition of pyruvate, sucrose or N A D H . The activation energy decreased by 10 ~o in the urea solutions. There was a 20 ~ decrease in the activation energy after addition of 2 M NaC1 and a 34 ~ decrease after addition of 4 M NaC1. The time dependence of the changes on the enthalpy of denaturation after reaction with n-ethylmaleimide is shown in the bottom portion of Fig. 3. When 2.2.10 -4 M n-ethylmaleimide is added, the ratio of sulfhydryl reagent to the total in sulfhydryl content of the lactate dehydrogenase is 1/4, and with 4.4.10- + M sulfhydryl reagent this ratio is 1/2. The total number of sulfhydryl groups in lactate dehydrogenase was calculated from the value in 8 M urea. The enthalpy of denaturation decreases after n-ethylmaleimide addition. At least 1 h at 25 °C is necessary for complete reaction. A concomitant decrease is seen in the ratio dH/dHeff. There was no change in the melting temperature after the sulfhydryl reagent addition.
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Fig. 3. Time dependence of the reaction of n-ethylmaleimide with lactate dehydrogenase. The ratio of the calorimetric heat to the effective enthalpy of activation is given in the upper l~ortion of the figure and the effect on the calorimetric heat in the lower part of the figure. ©, 2.2 × 10-4 M n-ethylmaleimide, 51 mg/ml protein, the ratio of sulthydryl reagent to protein sulthydryl is 1/4. n, 4.4 × 10-4 M n-ethylmaleimide, 51 mg/ml protein, the ratio of sulfydryl reagent to protein sulfhydryl is 1/2.
149 A comparison o f the effects produced by modifying lactate dehydrogenase with n-ethylmaleimide and with p-hydroxymercuribenzoate is shown in Fig. 4. In these experiments the protein samples were treated with the sulfhydryl reagents for 3--4 h at 4 °C before the calorimetric measurements. The melting temperature is shown at the top o f Fig. 4. With b o t h sulfhydryl reagents there was no change in the Tm when half the sulfhydryl in the protein were titrated. There was only a very small change in Tm (0.7 °C) at full sulfhydryl titration. The enthalpy changes are shown in the b o t t o m o f Fig. 4. The enthalpy o f denaturation decreased 30-60~o after reaction with n-ethylmaleimide, and 70-85
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Fig. 4. Changes due to modification of the sulfhydryl in lactate dehydrogenase (LDH) by n-ethylmaleimide or p-hydroxymercuribenzoate. The protein was assumed to contain 16 sulfhydryl groups/ mol (from titration in urea). The reaction mixture was incubated 4 h at 0 °C before the thermograms. The reaction mixtures contained 20-54 mg/ml protein and the sulfhydryl reagent concentrations were varied to give the appropriate ratios. Average values from six determinations. For the Tm plot: O, n-ethylmaleimide; [], p-hydroxymercuribenzoate, For the AH/A Heft plot : O, n-ethylmaleimide; II, p-hydroxymercuribenzoate. For the AH, dHerr and Ea plot: n-ethylmaleimide: AH, ©, Ancrr, V, E~,- ........ • p-Hydroxymercuribenzoate: AH, II, AHefr; 0, E.,, -........ .
150 after the p-hydroxymercuribenzoate treatment. AHeff also decreased after sulfhydryl modification of the protein, but the changes were of a lower order of magnitude than the changes in AH. As a consequence of these relative changes, the ratio AH/AHeff (shown in the middle portion of Fig. 5) decreased from a ratio of 1.0 ± 1 to 0.7 ~ 0.1 with n-ethylmaleimide and to 0.4 ~ 0.1 with p-hydroxymercuribenzoate. The changes in the activation energy are also shown in the bottom of Fig. 4. There is no change at 1/4 sulfhydryl titration. At full sulfhydryl titration the activation energy is decreased by 35 ~ and the values are similar to the value for lactate dehydrogenase in 4 M NaC1. The calorimetric experiments were at high protein concentration. (Required because of the small volume of the calorimetric cells). At these high protein concentrations there was no observable dissociation of the lactate dehydrogenase at 25 °C even at full titration of the sulfhydryl groups. The sedimentation measured by ultracentrifugal analysis was identical before and after full sulfhydryl titration. The protein appeared at the top of the 5 ~ acrylamide gels and there were no additional bands after full sulfhydryl titration. The gels were heavily loaded and any monomeric lactate dehydrogenase would have appeared in the middle of the gels. DISCUSSION The enthalpy of thermal denaturation of lactate dehydrogenase is 430 kcal/mol from a linear extrapolation to 0 °C/min heating rate. This value is considerably higher than the values previously reported for simple globular proteins [3, 4, 6, 10-12, 17] and for avidin [16]. This relatively high value for the enthalpy of denaturation of lactate dehydrogenase simply reflects its higher molecular weight. If the enthalpy change is expressed in calories/g, the value for lactate dehydrogenase is lower than the values reported for the simpler globular proteins. The enthalpy of denaturation of lactate dehydrogenase per tool of subunit (107 kcal/mol at 0 °C/min heating rate) is of the same order of magnitude as the value for lysozyme which has approximately the same degree of secondary structure. Hence there does not appear to be any large contribution to the total enthalpy of denaturation of lactate dehydrogenase to the interactions between subunits. It is possible that interactions between subunits in a protein does not in general, produce any large contributions to the total enthalpy of denaturation. Donovan and Beardslee [17] found that the melting temperature increased for the trypsin, soybean trypsin inhibitor complex and the trypsin, ovomucoid complex compare to the individual proteins, but the enthalpy of denaturation was essentially additive. In the denaturation of lactate dehydrogenase, both dissociation into subunits and loss of secondary structure in the monomer units should occur. Only one transition was observed. It is possible that the denatured state in lactate dehydrogenase is the denatured tetramer. Ribonuclease, lysozyme and chymotrypsinogen all have been reported to retain some degree of ordered structure after heat denaturation [28]. An alternative possibility is that dissociation is not a rate-limiting step and that the observed heat includes both loss of secondary structure and dissociation of subunits. As in the ease of the enthalpy of thermal denaturation, the activation energy of the lactate dehydrogenase denaturation is considerably higher (172 kcal/mol) than values reported for simpler proteins. Crescenzi and Delben [5] used the Beech method for calculating activation energies and obtained values of 90 kcal/mol for ribo-
151 nuclease, 101 kcal/mol for lysozyme and 80 kcal/mol for chymotrypsinogen A. Donovan and coworkers [16, 17] also used the method of Beech and found values of 69 kcal/mol for soybean trypsin inhibitor, 63 kcal/mol for trypsin, and 90 kcal/mol for avidin. The high value for the activation energy for lactate dehydrogenase probably reflects its high molecular weight as the value considered per mol of monomer is lower than the values for the simpler proteins. The small change in activation energy of lactate dehydrogenase in solutions containing urea is similar in magnitude to the changes reported for ribonuclease and lysozyme at low urea concentrations [5]. The very much larger changes in activation energy of lactate dehydrogenase in concentrated NaC1 solutions and for the sulfhydryl-modified protein correspond in magnitude to the change in the values obtained on addition of guanidine. HC1 to ribonuclease or lysozyme [5]. The very small changes in Tm, and E a for the thermal denaturation of freshly prepared protein at 10 °C/min in 1 and 2 M urea are in general agreement with previous reports that urea cleaves lactate dehydrogenase into subunits only at high concentrations [24]. However, the changes observed at the lower heating rate suggest that there is a slow modification by low concentrations of urea, and the modifications are both time and temperature dependent. Sucrose is well known to stabilize proteins. The stabilization of lactate dehydrogenase by sucrose is most noticeable in the increased Tm ( + 7.6 °C) while there is no change in the enthalpy. Sucrose appears to prevent the onset of denaturation while not affecting the mechanism of denaturation. There have been numerous reports of the stabilization of lactate dehydrogenase by cofactor binding [18]. Hence, stabilization against thermal denaturation was expected and observed. Since the protein melts at a higher temperature, some increase in A H due to the higher temperature is expected. However, the increase in the Tm in the presence of NADH (5 °C) is less than the increase in the presence of sucrose (8 °C). Within experimental error (4-42 kcal/mol) there is no increase in A H in the presence of sucrose. Hence the effect on A H of the increased Tm in the NADH solutions is expected to be small and the degree of stabilization of lactate dehydrogenase against thermal denaturation by its cofactor NADH appears to be directly related to the enthalpy of binding of the cofactor. From an extrapolation of the calorimetric data of Hinz and Jaenicke [23] from 40 °C, the value for AHbt,ding of N ADH to lactate dehydrogenase would be of order of magnitude --90 to --120 kcal/ mol at 71 °C. A lower estimation can also be made from the kinetic data of Borgmann et al. [29]. However, the interpolation from this data is more difficult since equations for variation of ZlHblndlng with temperature were not given. An extrapolation of the calorimetric data for NAD binding to glyceraldehyde-3-phosphate dehydrogenase [30] to 71 °C would give a value of --140 kcal/mol for four NAD bound. This suggests the binding of the cofactor, either N A D H or NAD, is strongly exothermic and of comparable order of magnitude with proteins requiring this cofactor. With lactate dehydrogenase, the increase of 85 kcal/mol in AH in the presence of NADH can easily be accounted for by the dissociation of bound NADH from the protein. The situation is quite different from the binding of biotin to avidin [16] where stabilization is very much greater than expected from the heats of binding of biotin to avidin, and change in heat capacity were postulated to explain the very high degree of stabilization. The stabilization of lactate dehydrogenase to thermal denaturation in the
152 presence of its cofactor N A D H , and the absence of any stabilization by the substrate pyruvate could be due to binding of substrate without any concomitant conformational change in the protein as occurs with cofactor binding, or to very weak binding of substrate. However, these data do suggest that binding of substrate may occur only after binding of cofactor. This latter suggestion has been made previously as part of the proposed mechanism o f enzymatic action [31, 32]. The interpretation of the thermal behaviour of lactate dehydrogenase after modification of sulfhydryl groups appears to be complex. The melting temperature after sulfhydryl modification is not decreased as is the case after urea or NaCI addition to the enzyme solution. Titration of sulfhydryl groups might have been expected to affect the dissociation of subunits and hence the Tin, since the dissociation and reassociation of tetramer at low protein concentrations is dependent on the concentration of free sulfhydryl groups and protective agents such as dithiothreitol increase the reassociation [19]. The disc gel electrophoresis and the ultracentrifugal data clearly show that the initial state is still tetrameric. It is not known whether the final thermally denatured states are the same with and without sulfhydryl modifications of the lactate dehydrogenase. Some changes in enthalpy are expected because of modification of the tetrameric initial state. Differences in cooperativity between subunits would also be expected because of the proximity of the sulfhydryl groups to the sites of association between monomers [18, 19]. The changes in Tm suggest that dissociation into subunits may occur in the NaC1 solution, or in the urea solution on standing, but that such dissociation is not a major factor in the sulfhydryl-modified protein. The higher magnitude of the change in d H with p-hydroxymercuribenzoate to n-ethylmaleimide modification could be due to differences in accessibilities of the protein binding sites to these reagents. However, in both sulfhydryl modifications the enthalpy of thermal denaturation is decreased while Tm change is very small. Privalov and Khechivashvili [12] have shown that if it is assumed that the denaturation represents a two-state transition to which the van't Hoff equation is applied, an effective enthalpy can be calculated at the midpoint of the transition. For a two-state reversible transition this effective enthalpy would be the van't Hoff enthalpy. The ratio of the calorimetric enthalpy to the effective enthalpy would be equal to 1 for a two-state transition, greater than one if there is a significant population of intermediate denatured states and less than one if there are non-equilibrium conditions in the cell due to irreversible denaturation. Since the total denaturation of lactate dehydrogenase is irreversible, values of AH/AHe, less than one are predicted. Values less than one are found in the presence of high concentrations of NaC1 and on standing in urea and for the sulfhydryl-modified lactate dehydrogenase. However, for the pure lactate dehydrogenase (and with the pyruvate and sucrose additions) the ratio of dH/dHeff is 1.0 ~ 0.1. In these cases values less than one also should be found since the total denaturation is irreversible when the protein is heated to 70 °C. The observed values of 1.0 ± 0.1 could be fortuitous, and the actual values less than 1.0 within the limits of experimental accuracy. However, this observed value may reflect the reversible nature of the denaturation below Tin. At temperatures above Tin, the reversibility could critically depend on factors such as temperature of heating, time of heating and cooling, and exchange or oxidation of sulfhydryl groups. The precipitation which occurs after heating to 70 °C and cooling and reheating suggests that some aggregation has occurred. The observed value of 1.0 suggests that the two-
153 state d e n a t u r a t i o n m o d e l for lactate dehydrogenase m a y be a p p r o p r i a t e u n d er specified conditions. ACKNOWLEDGEMENT S u p p o r t f r o m grants f r o m the N a t i o n a l R e s e a r c h C o u n c i l o f C a n a d a and the A l b e r t a H e a r t F o u n d a t i o n are gratefully acknowledged. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
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