Biocon)ugate Chem. 1901, 2,309-310
309
ARTICLES Catalytic Activity and Thermostability of Dehydrogenase Conjugates with Cortisol and Progesterone’ Dmitriy I. Metelitza,’ Alexandr N. Eryomin, Elena I. Karasyova, and Vera L. Markina Institute of Bioorganic Chemistry, BSSR Academy of Sciences, 220045, Minsk, U.S.S.R. Received September 17,1990 The conjugates of glucose-6-phosphate dehydrogenase, lactate dehydrogenase, and malate dehydrogenase with progesterone and cortisol, containing 1-40 steroid molecules per enzyme molecule, were obtained by the reactions of N-succinimide esters of the 3-[0-(carboxymethyl)oxi~nes)]of cortisol and progesterone with a protein in a water-DMFA (10%) medium. The catalytic activity and thermostability of dehydrogenases and their steroid conjugates were kinetically studied. The effects of the modification degree on the activity and thermostability of dehydrogenases caused by their hydrophobization were studied and discussed. Practical recommendations for using the dehydrogenase-steroid conjugates in enzyme immunoassay are given.
INTRODUCTION Modern applied enzymology solves problems via two main directions: the first is biocatalyst engineering, which involves modification of a biocatalyst itself by using genetic, physical, and/or chemical methods; the second is medium engineering which involves modification of the microenvironment of a biocatalyst, either by introducing additives or solid matrices into an elaborated medium or by varying the composition of the liquid medium itself (I, 2).
The practical requirements of biotechnology and basic research often join both foregoing approaches into one to solve any task, for instance, in enzyme immunoassay for a number of antigens ( 3 , 4 ) . In this case it is necessary to obtain the enzyme conjugates with water-insoluble antigens in the water-organic media in order to retain the enzyme catalytic activity and the immunochemical reactivity of the bound antigens in a maximum degree (3-5). The high hydrophobicity of some antigens, e.g. steroids, aromatic carcinogens, toxicants of environmental media, etc., causes the necessity of finding methods for enzyme immunoassay in organic media. We believe that enzyme immunoassay in reversed micelles of surfactants in organic solvents may be especially hopeful (6-8). Recently we have shown the principal possibility of the interaction of an antigen (peroxidase) with antibodies (anti-peroxidase) in the reversed AOT (sodium bis(2-ethylhexyl) sulfosuccinate) micelles in heptane (9). In 1989,we developed the homogeneous enzyme immunoassay of progesterone in the mixed reversed micelles of AOT and Triton X-45 in heptane using horseradish peroxidase as antigen label (IO). During the past 5 years, we have carried out a systematic kinetic study of the changes in the enzyme catalytic activity 1 Abbreviations used: DMFA,dimethylformamide;GP, glucose &phosphate,GGPDH,glucose-6-phosphatedehydrogenase;LDH, L-lactate dehydrogenase; MDH, L-malate dehydrogenase; COR, cortisol; 3-COR,cortisol 3-[0-(carboxymethyl)oxime]N-succinimide ester; PROG, progesterone; 3-PROG, progesterone 340(carboxymethyl)oxime]N-succinimide ester; TNBS,trinitrobenzenesulfonic acid; EIA, enzyme immunoassay.
* To whom correspondence should be addressed: Professor Dr. D. I. Metelitza, Institute of Bioorganic Chemistry, BSSR Academy of Sciences, 512 Zhodinskaya, 220045, Minsk-45,
U.S.S.R.
and thermostability that resulted from their modification with cortisol, progesterone, and strophantin. In the present paper we summarize and discuss the effects of the chemical modification with cortisol and progesterone in water-organic solvents on catalytic activities and thermostabilities of three dehydrogenases having quaternary structure. Table I lists the characteristics of the dehydrogenases used and degrees of their modification, “n”.We would like to especially note that all the enzyme steroid conjugates were successfully applied by us in enzyme immunoassay. EXPERIMENTAL PROCEDURES Reagents. We used glucose-6-phosphate dehydrogenase from Leuconostoc sp 5, (NPO Ferment, Vilnius, U.S.S.R.) with a specific activity of 350-400 IU/mg and L-lactate and L-malate dehydrogenase from pig heart mitochondria (Reanal, Hungary) with specific activities of 150 and 180IU/mg, respectively. As emzyme substrates, we used glucose6-phosphate and NADP+ (Reanal),sodium oxalate and pyruvate (Sigma, St. Louis, MO), and NADH (Reanal). Cortisol and progesterone were purchased from Germed (FRG), and Sephadex G-25 was from Pharmacia (Uppsala, Sweden). N-Succinimide esters of the 3-[0(carboxymethyl)oxime] of cortisol and progesterone were obtained according to ref l l a with some modifications and kindly provided by Dr. V. D. Matveentzev (Institute of Bioorganic Chemistry, Minsk, U.S.S.R.). DMFA, ethanol, 1,4-dioxane, propanol, heptane, and octane were distilled before use. All other reagents were from Reachim (U.S.S.R.). Preparation of Dehydrogenase Conjugates with Cortisol and Progesterone. The dehydrogenase conjugates with steroids were obtained in water-DMFA solutions of various compositions by a known method (11) with some modifications (12). We performed the reaction of 3-PROG or 3-COR with dehydrogenases at a modifier/ enzyme molar ratio ranging from 10 to 100. The reaction was usually carried out at 25 “C in a NaHC03 buffer (pH 8.35) containing 10% of DMFA for 2 h. The modifier (3-COR, 3-PROG) stability is determined by pH value and strongly decreases at pH 9 and higher. At pH 8.35 the modifiers reacted with proteins much faster than their hydrolysis occurred, since the hydrolysis rate of modifiers at pH 8.35 was very slow.
1043-18Q2f91129Q2-Q3Q9$Q2.5Qf Q 0 1991 Amerlcan Chemlcal Soclety
910
Metelltza et al.
Bloconlugete Chem., Vol. 2, No. 5, 1991
Table I. Dehydrogenases Modified by Progesterone and Cortisol MW, subunit modification enzymes EC kDa number degree 4 2-40 L-lactate dehydrogenase 1.1.1.27 140 (LDH) L-malate dehydrogenase 1.1.1.37 70 2 3-20 (MDH) 2 1-35 gluccae-&phcaphate 1.1.1.49 104 dehydrogenase (GGPDH)
100
w A + d d + 4
50
0
10 20 Concentration of organic solvent, 16
Figure 2. Effects of organic solvents on the initial catalytic activity of G6PDH: 1, DMFA;2, propanol; 3, glycerol; 4, ethanol; 5 , l,4-dioxane.
0 -
1
1
2 50 W8Ye
L
3 00 legth,
350
Figure 1. The absorption spectra of initial LDH (1) and its conjugate with cortisol (2) and the difference spectrum of conjugate LDH-3-COR-16 (3) at the concentration of 3.0 mg/
mL.
Unreacted modifiers were removed by dialysis. The full absence of free modifiers and their hydrolization products in the conjugate samples was spectrophotometrically proved after the dialysis procedure. For characterization of the conjugates, their difference absorption spectra were monitored versus the same concentrations of nonmodified proteins (see Figure 1).The numbers of the enzyme-bound modifier molecules "n" (or modification degree) were calculated from the intensity of the difference spectra of the conjugates at 250 nm using the molar extinction coefficient 16 000 M-' cm-I for COR (PROG) (13). The modification degree for the three dehydrogenases varied from l to 40 (see Table I). The conjugates of G6PDH with 3-PROG, containing 3,5,7,15, 20, and 35 molecules of 3-PROG and abbreviated as GGPDH-3-PROG-3 GGPDH-3-PROG-35, were studied in detail as well as the conjugates of LDH with 2,20, and 40 3-PROG and conjugates of MDH with 3,15,20, and 28 3-PROG, referred to as LDH-3-PROG-2 LDH-3-PROG40 or MDH-3-PROG-3 ...MDH-3-PROG-28. The conjugates G6PDH-3-COR-25, MDH-3-COR-8, and some others were prepared and studied in detail. All the obtained conjugates were stored in 0.1 M phosphate buffer, pH 7.4, at 4 "C. Determination of the Catalytic Activity of Dehydrogenases and Their Conjugates. The catalytic activity of the G6PDH and its conjugates was characterized by the GP dehydrogenation in 0.1 M NaOH-glycine buffer (pH 9.1) containing 0.4 mM NADP+, 1.0 mM GP, and, as a rule, 500 ng/mL of a biocatalyst at 20 "C (14). The enzymatic activities of LDH, MDH, and their conjugates were characterized by the initial rates of pyruvate or oxaloacetate reduction in 0.1 M phosphate buffer, pH 7.5, containing 0.1 mM NADH and 0.5 mM pyruvate or ox-
...
...
aloacetate, respectively, a t 20 "C (15). Both the reactions were spectrophotometrically monitored by using the absorption decrease of NADH at a wavelength of 340 nm (t = 6220 M-I cm-I). In all the cases, the linear dependence of the optical density alteration on the reaction time was observed which permitted the correct calculation of the initial reaction rate in M s-l. Thermoinactivation of Dehydrogenasesand Their Conjugates with Steroids. In a temperature range of 35-47 "C for G6PDH and 38-47 "C for its conjugates with progesterone, the biocatalyst thermoinactivation was studied in the twice-distilledwater at various initial protein concentrations. Thermoinactivation of two other dehydrogenases and their conjugates was studied at 20-45 "C in the twicedistilled water (LDH, LDH-3-PROG-19, MDH, MDH3-PROG-16) or 0.1 M phosphate buffer, pH 7.5 (LDH, MDH, and MDH-3-PROG-24). The enzyme or its conjugate solutions with varying protein concentrations were kept at different temperatures and aliquota were taken for determination of the enzymatic activity under the standardizated conditions at 20 "C as described above. The thermoinactivation of the dehydrogenases was characterized by the effective first-order rate constants, kin, which were calculated from the semilogarithmic anamorphoses of kinetic curves of the enzymatic activity decrease, log (A/Ao) - t, where A0 and A are the catalytic activities of the native and partially inactivated biocatalyst, respectively, and t is the reaction time. All the spectral measurements were carried out in the thermostated cuvettes of Specord UV VIS or Specol 21 instruments (Karl Zeiss, Germany). RESULTS AND DISCUSSION
The Environment'sEffects on the GGPDH Catalytic Activity. At the preparation of the dehydrogenase conjugates with steroids, it is necessary to add the organic solutions of the water-insoluble modifiers 3-COR and 3-PROG into the buffered enzyme solutions. Therefore, it is very important to know how the organic cosolvent acts on the enzyme itself. As shown in Figure 2, the increasing concentrations of propanol, ethanol, or 1,4-dioxane decrease the G6PDH catalytic activity while DMFA concentrations below 8% increase it (dependence 1). In the futher experiments we
ActMty and Thermostability of
0 '
l
20
Dehydrogenase Conjugates
Bioconjugate Chem., Vol. 2, No. 5, lQQl 311
I
I
40
60
Temperature,
OC
Figure 3. The number of free amino groups of the lysine residues of GGPDH determinedby protein titration with TNBS at various temperatures: 1,initial enzyme; 2, GGPDH treated with a waterDMFA mixture for 1 h. used the mixtures of the buffered solutions with DMFA (10%) as a solvent that permits retention of a high enzymatic activity of the prepared conjugates. The stimulating action on the GGPDH catalytic activity of another bipolar solvent, dimethyl sulfoxide, was shown earlier (16). Optimization of the Synthesis of Dehydrogenase Conjugates with Steroids. The modification degree of dehydrogenases by the 3-COR and 3-PROG agents is determined by the enzyme structure (a number of free amino groups), the medium composition (the DMFA content), the modifier/enzyme ratio, the temperature, and the reaction time. I t is well-known that 3-COR and 3-PROG effectively react with free lysine amino groups (5, 11). Each LDH subunit contains 24 lysines (17),i.e. the maximum modification degree of this enzyme (n = 96) may be achieved. When GGDPH was kept in a solution containing 10% DMFA, the total number of amino groups accessible for such an agent as TNBS significantly increased (see Figure 3). At 25 "C the number of such groups equaled 40, while only 22 such groups were available in the initial enzyme in a buffered solution. Noteworthy is that at 45 "C the difference between the initial enzyme and that kept in a water-DMFA (10%) mixture disappeared. The maximum modification degree that the GGPDH may achieve is n = 80. Thus, the temperature increase causes a growth of the number of the free amino groups number which are accessible for the modifier but simultaneously the enzyme inactivation rate increases. Therefore, all the conjugates were prepared a t 20-25 "C. A growth of the modifier/enzyme ratio increases the modification degree of three dehydrogenases (Figure 4). For the GGPDH and LDH modification by progesterone, the linear dependences of "nuon the [3-PROG]/ [enzyme] ratio were obtained. The modification degree and activity are determined by the reaction time, while an inactivation of enzymes and their conjugates is dependent on their contact with organic cosolvent. The optimum combination of the modifierlenzyme ratio, the medium composition, and the reaction time permit us to obtain highly active conjugates of the dehydrogenases with cortisol and progesterone. Figure 5 shows the dependence of the conjugate catalytic activity on the modification degree of dehydrogenases. At a high modification degree the conjugates LDH-3-PROG were found to retain the catalytic activity of the initial enzyme. The conjugates MDH-3-COR and MDH-3-PROG with n
100
50
[Modifier] / [Enrqme]
Figure 4. The dependence of the modification degree (n)on the initial [modifier]/ [enzyme] ratio for MDH with cortisol (1)and progesterone (2), LDH with cortisol (3) and progesterone (4), and GGPDH with progesterone (5). 140 bp
+A d
d P
+
s
100
I 0
1
I
20
40
Yodif icat i o n degree
Figure 5. The dependences of catalyticactivityof the conjugates of dehydrogenases on modification degree by cortisol and progesterone: 1,MDH-&COR; 2, MDH-3-PROG; 3, LDH-3-PROG; 4,LDH-3-COR; 5, GGPDH-3-COR; 6, GGPDH-3-PROG.
= 16had an activity that was 1.2-1.5-fold higher than that of nonmodified protein. The GGPDH modification resulted in a considerable loss of its catalytic activity. This loss increases with the growth of modification degree of LDH (Figure 5 ) or GGPDH (Table 11). As seen from Table 11, growth of the modification degree leads to an increase of the Michaelis constants for the substrate (GP) and cofactor (NADP) which is also unfavorable for the enzymatic catalysis. The optimum conditions for the conjugate preparation allow achievement of a very high degree of protein modification: in LDH 42% of free amino groups and in GGPDH 44 % of such groups were modified (see Figure 5 and Table 11). I t is clear that at such high modification degree of the enzyme, complete retention of its catalytic activity could hardly be expected. We believe its level equal to 50-70 % of the initial enzyme activity is sufficiently high. Interesting results were obtained at the MDH modification by 3-COR and 3-PROG (Figure 5, curves 1and 2): the catalytic activity of conjugates MDH-3-COR-8 and
312
Metelitre et al.
Bioconlugete Chem., Vol. 2, No. 5, 1991
Table 11. The Catalytic Activity of Glucose-6-phosphate Dehydrogenase and Its Conjugates with Progesterone activity vs that of G6PDH and its conjugates WK,, M for GP 108 K,, M for NADP+ initial G6PDH G6PDH treated with DMFA (10%) 2.3-2.5 100 GGPDH in buffer solution 4.2-4.7 100 2.4-3.5 76-85 3.9-4.7 G6PDH in buffer-DMFA mixture 83 3.4 98 GGPDH-3-PROG-3 4.2 82 3.0 97 GGPDH-3-PROG-10 4.3 61 3.3 75 GGPDH-3-PROG-15 7.7 55 4.7 68 G6PDH-3-PROG-20 10.5 47 4.2 58 G6PDH-3-PROG-25 10.5
I
20
I
L
40
60
I
20
40
60
40
20
60
Z, min
Figure 6. The semilogarithmic anamorphoses of the kinetic curves of the catalytic activity loss for MDH (a), control MDH (b), and conjugate MDH-3-PROG-16 (c) in buffered solution at 40 O C : 1, 0.005; 2, 0.010; 3,0.050; 4,O.lO mg/mL of protein. MDH-3-PROG-15 equals to 149 and 111% ,respectively, relative to that of the initial enzyme. Thermoinactivation of the Dehydrogenases and Their Conjugates with Steroids. Figure 6 shows the semilogarithmic anamorphoses of kinetic curves of the catalytic activity loss a t 40 O C for the initial MDH (a), the control enzyme incubated in the water-DMFA (10%) mixture (b),and conjugateMDH-3-PROG-16 (c). As seen, the biocatalyst inactivation at its various initial concentrations ranged from 0.005 to 0.1 mg/mL and is described by the first-order equation below, which gives a fair approximation for inactivation of two other dehydrogenases (GGPDH, LDH) and their numerous conjugates at various concentrations and different temperatures (1822). In all the cases, the rate constants of the biocatalyst inactivation, kin, were strongly dependent on the initial protein concentration. These dependences for GGPDH, LDH, MDH, and numerous conjugates of LDH and MDH are described byequation 1,connecting ki, with the initial kin
= ko/(1
+ Ako[Elo)
(1)
enzyme concentration, [El0 (18-22), where ko is the rate constant of the protein inactivation at its infinite dilution, A is the reverse rate in M-’ s and dependent on temperature, and [El0 is the initial concentration of subunit enzyme or its conjugate. Transforming eq 1as shown in Figure 7, one can calculate the value of the constants ko and A for the inactivation of dehydrogenases and their conjugates. Table I11 lists the ko values at 40 O C for various forms of LDH and MDH and their conjugates with progesterone. As seen from this table, the inactivation rate of LDH and MDH in buffered solution are significantly less than in water, i.e. the buffer salts stabilize both dehydrogenases. The MDH modification by 3-PROG results in some enzyme stabilization, while the LDH modification under the same conditions decreases the enzyme stability by 1.6-fold.
The experimental data obtained by us (18-22) support the assumption that the dissociative inactivation of dimeric (or tetrameric) dehydrogenases may be described by the following simplest scheme: ki
E, * 2E kz
-. kd
2Ed
(2)
where E2 is a dimer, E is a monomer (subunit), k1 is the rate constant of the dimer dissociation, k2 is the rate constant of the monomer association, and kd is the rate constant of the monomer denaturation. As it was shown by us earlier (18-22) the rate constants ko calculated with eq 1were close to the rate constants of the dimeric dehydrogenases dissociation, kl (see eq 2);i.e. the biocatalyst dissociation may be the limiting stage of the enzyme thermoinactivation. The temperature dependence of the rate constants, ko, for the G6PDH inactivation is described by the Arrhenius plot (Figure 8), from which the activation energy for the thermal dissociative inactivation of this enzyme was calculatedtobe equal to 109.7 kcal/mol. For a comparison, from the temperature dependence of kin, the effectiveEnd’s were calculated for the thermoinactivation of G6PDH at various initial concentrations: G6PDH concn, mg/mL E&, kcal/mol infinite dilution
0.001 0.002 0.004 0.005
109.7 135.5 142.0 143.0 143
As seen from these data, increase of the G6PDH solution concentration results in the rise of End values. Modification of free amino groups of GGPDH gives the conjugates, the hydrophobicity of which increases with the growth of the modification degree. The G6PDH modification by steroids is always accompanied by an increase in the effective rate constant of the modified
Bloconlugete Chem., Vol. 2, No. 5, l9Sl
Actlvity and Thermostability of Dehydrogenase Conjugates 1O2 *
0
313
bH],mg/ml 4
2
4
3 \O
it
2
\
8 1
3,1 I
0'
095
lo2-
312 103.~-',
I
1,o
EDHI,-/la1
Figure 7. The dependences of the reverse effective inactivation rate constants for the different forms of MDH (a) and LDH (b) at 40 O C on the initial concentration of the biocatalysts: 1, initial enzyme; 2, control enzyme; 3, conjugates MDH-3-PROG-16 (a) and LDH-3-PROG-19 (b).
IC-'
Figure 8. The temperature dependence of G6PDH inactivation rate constant in very diluted solution ko calculated by using eq 1.
Table 111. The Inactivation Rate Constants for Various Forms of MDH, LDH, and Their Conjugates at 40 O C Calculated from Experimental Data by Using Equation 1 biocatalyst, medium 109 k,, s-1 LDH, 0.1 M phosphate buffer, pH 7.5 0.13 LDH, water 2.00 L D H m ~ water , 3.33 LDH-3-PROG-19, water 3.33 MDH, 0.1 M phosphate buffer, pH 7.5 MDH, water MDH,,*l, water MDH-3-PROG-16, water
0.76 1.67 1.54 1.43
enzyme inactivation kin as compared with its value for the initial enzyme (see Table IV). It should be mentioned that for conjugates GGPDH3-PROGI unlike the enzyme itself and LDH, MDH, and their conjugates with progesterone, a simple dependence of ki,on the initial protein concentration was not obtained; i.e. eq 1was not fulfilled in this case. The dependences of ki,on the protein concentration for conjugate GGPDH3-PROG-35 at 38,45, and 47 O C are compared in Figure 9. These dependences are complex and have maximums, the positions of which are determined by temperature. The complicated dependence of ki, on the GGPDH concentration does not allow the calculation the ko values for this conjugate inactivation a t various temperatures. The temperature dependences of the effective rate constants kin for GGPDH a t various concentrations (0.001, 0.005,0.010, and 0.020 mg/mL) are described by the Arrhenius equation and characterized by the same value of Eaet equal to 109.7 kcal/mol. Table V lists the thermodinamic activation parameters calculated for the process of catalytic activity loss by GGPDH and its conjugate GGPDH-3-PROG-35 a t 45 OC. Modification of this
0,005r 0 0
I
0,Ol
I
I
I
0,02
Os03
0.04
~ ~ P D H - ~ - P R w/al oo-~~~
Figure 9. Effect of the initial concentration of conjugate G6PDH-3-PROG-35 on the effective values if the inactivation rate constants at 38 (a), 45 (b) and 47 "C (c).
enzyme does not change the activation parameters of its inactivation for such a process in strongly diluted solutions. Increase of the modification degree of GGPDH is accompanied by gradual alterations of the character of dependence"ki,- protein concentration". Figure 10shows such dependences at 45 "Cfor initial GGPDH, the control enzyme, and GGPDH conjugates with increasing progesterone content. As seen from Figure 10, ki, of GGPDH decreases with increasing enzyme concentration. The protein modification results in the reversed dependences that have been clearly shown for the conjugates of GGPDH
314
Bloconjugete Chem., Vol. 2, No. 5, 1991
Table IV. Thermoinactivation Rate Constants for GGPDH and Its Conjugate GGPDH-3-PROG-36 (4.lw, 8-l) at Various Temperatures and Protein Concentrations
temp, "C 35 40 45 47
0.001 0.08
1.60
0.002 0.06 0.79
81.5
30.5
88.3
66.2
GGPDH, mg/mL 0.003 0.004 infinite dilution 0.20 0.04 0.03 5.00 0.36 0.28 52.0 19.7 19.0 37.0 141.0 43.7
G6PDH-3-PROG-35, mdmL 0.005 0.010 0.020 0.030 0.040 1.30 1.30 1.60 1.10 1.20 3.30 5.10 79.0 50.6 16.0 77.0 73.0 220.0 240.0 190.0 140.0 290.0
_ _ _ _ _ _ _ ~ ~
temp, "C 38 40 45 47
0.001 0.50
6.30 53.0 150.0
with 7 and 35 molecules of 3-PROG. The ki, for these conjugates increases with the growth of the protein concentration up to 0.020 mg/mL. There are two possible explanations: first, the subunit protein is inactivated due to its dissociation into monomers; second, the same protein may be inactivated because of its "excessive" association. The increasing hydrophobicity of GGPDH conjugates results in the formation of the large aggregateswhich causes a loss of the protein activity with the increase of its concentration up to 0.02 mg/mL (see Figure 10b,c). As seen from Table 111,the LDH modification with progesterone slightly decreases the enzyme stability, while the MDH modification increases the enzyme stability below 30 "C (see Table VI), but above this temperature the thermostabilities of the initial and modified MDH are similar. Commenting on the data listed in Table VI, it should be emphasized that only the comparison of kovalues is correct since the biocatalyst concentration can influence the kkvalues of the enzyme and its conjugates in absolutely different ways. It is very noteworthy that the changes in the catalytic activity and thermostability of these dehydrogenases after their modification by steroids are ppallel. Owing to modification of LDH by 3-40 molecules of 3-PROG, its activity decreases up to 86% of the initial level, while at the moderate modification of MDH (3-15 molecules of 3-PROG) the biocatalyst activity increases up to 111% , which corresponds with an increase of thermostability of the modified enzyme (see the kin values in Table 111). Increase of MDH modification (n = 20-28) results in decrease of the catalytic activity of this enzyme (Figure 5). The parallel change of catalytic activity and thermostability was found for GGPDH and its conjugates. The modification results in decrease of the catalytic activity (Figure 5, Table 11)and a loss of thermostability (Figure 10,TableIV). The parallel changes of the enzyme activity and stability are often observed in applied enzymology (23). In our opinion, change of the catalytic activity and thermostability of the dehydrogenase due to the modification appears to be connected with two main reasons: firstly, the DMFA action on the enzyme and, secondly, the enzyme hydrophobization owing to the insertion of modifiers, 3-COR or PROG, in its molecule. The contribution of the former factor is absolutely obvious from Figure 11: a loss of the GGPDH activity results from the enzyme incubation in DMFA-containing solutions. For instance, at 25 "C in the water-DMFA (30%) mixture GGPDH completely losses its activity after a 1-h incubation. A Comparisonof the activities of the native and modified GGPDH (see Table 11)indicates that they are significantly different. The enzyme activity decreases for 1 5 2 4 % due to the DMFA action on enzyme. Increase of the degree
Metelltza et al.
of the protein modification decreases its activity in such a manner that the modifier itself decreases the activity value by 11-15% as compared with that of the control enzyme treated with the mixture of water with DMFA; i.e. the enzyme activity is supressed not only by an organic cosolvent but also by modifier itself. Modification results in decrease of the thermostability of the GGPDH conjugates as well (Table IV). As seen from Table 111, the overall LDH stability after its modification decreases by 67 % as compared with that of the control and initial protein; Le. this dehydrogenase undergoes all the significant structural changes as a result of the DMFA action and then does not alter its stability owing to an insertion in its molecule of 19 modifiers. The direction of the stability changes of modified LDH and MDH (as well as their catalytic activity) are quite opposite. Increase of the MDH activity and stability as a result of its modification by progesterone may be accounted for a hydrophobization of some free amino groups of the lysine residues responsible for the interaction between the MDH subunits. Presence of such lysine residues in MDH is well-known (24). Neutralization of the positive charge of the MDH as well as LDH groups by their acetylation reduces the enzyme stability ( 17, 24). Hydrophobization of such residues stabilizes, probably, the quaternary structure of MDH. The known data show that the hydrophobization of the internal sites of a protein stabilizes it, while the hydrophobization of the enzyme surface destabilizes it in the buffered solutions (25-27). What are the mechanisms of the action of DMFA and the modifiers, 3-COR and 3-PROG, on the dehydrogenases? Before giving an answer to this question, it should be mentioned that LDH and MDH easy dissociate into their subunits, which are inactive for LDH, while the immobilized MDH monomers have the same catalytic activity (28-30). The GGPDH subunits have much lower activity than that of the enzyme with undisturbed quaternary structure (31, 32). I t was assumed that the quaternary structure of GGPDH is supported by two molecules of NADP+ (32, 33). However, Yoshida (34) proposed a hexameric structure for GGPDH containing a t least six structural NADP+ molecules. The DMFA action on the dehydrogenases and their conjugates causes a solubilization of the enzyme subunits; i.e. a protein losses the structural water which is substituted by the dipolar aprotic solvent (2). The moderate substitution of water for DMFA may enhance the enzyme activity as in the case of GGPDH (Figure 2), but increase of the organic cosolvent content causes a sharp decrease of the enzyme activity (Figure 11). Also, the initial action of DMFA on the dehydrogenase dissociation cannot be excluded. However, in our laboratory the data obtained which prove that the inactivating action of DMFA is realized by means of a solubilization of the enzyme subunits but not its dissociation into monomers, since the bifunctional agents (glutaraldehyde, dimethyl suberimidate, and dimethyladipimidate) did not preserve GGPDH from the inactivating action of DMFA (35). The effect of modification on the dehydrogenases involves a hydrophobization of internal and external sites of the enzyme (21,25-27). Modification of the external amino groups causes, as a rule, the destabilization of the enzyme, but its activity may be retained. At the modification of the internal amino groups, the enzyme, for instance MDH, may be stabilized and activated. At high modification degree the external amino groups react with a modifier which may inactivate a biocatalyst. Analysis of the temperature dependences of the effective rate constants for the dehydrogenase inactivation, kin, could not be overestimated, since the temperature course
Acthrity and Thermostability of
Dehydrogenase Conjugates
Bloconlugete Chem., Vol. 2, No. 5, 1991 315
Table V. Thermodynamic Activation Parameters for the GGPDH Inactivation at Various Concentrations and for Conjugate GGPDH-3-PROG-35 (45 "C) protein, mg/mL E&, kcal/mol AH*, kcal/mol AS*,kcal/mol O C AG*,kcal/mol G6PDH (infinite dilution) GGPDH, 0.001 mg/mL GGPDH, 0.005 mg/mL G6PDH-3-PROG-35,0.005 mg/mL
109.7 135.7 143.6 109.7
269.1 353.0 374.0 270.1
109.0 135.0 143.0 109.0
23.4 23.2 24.0 23.1
0
0
I 2
I
I
4
6
2, hourr
Figure 11. The dependences of the GGPDH activity on the DMFA content in a NaHCOa (2 5%) solution,pH 8.3 at 25 "C, and the enzyme concentration 1.5 mg/mL: 1,5; 2,lO; 3,20; 4 , 3 0 % DMFA.
0
Os03
0,02
0,Ol
[eotaq
0,04
, mg/ml
Figure 10. The dependencesof the effective inactivation rate constants at 45 O C for the initial GGPDH (a),enzyme treated by water-DMFA (10%) mixture (b), and the G6PDH conjugates containing3 (c),7 (d),and 35 (e) molecules of modifier 3-PROG. Table VI. The Effective Inactivation Rate Constants (16' h., a-1) for the MDH Forma and Its Conjugates (5.0 lo-' mg/mL) at Various Temperatures enzyme, medium temp, O C
20 25 30 35 40 45
MDH, buffer, pH 7.5 3.64 3.83 5.75 11.10 17.60 49.10
MDHm.trOl,
buffer, pH 7.6 2.04 2.30 3.07 3.96 8.82 33.70
MDH-3PROG-24, buffer, pH 7.5 1.05 2.30 2.56 2.68 9.58 29.50
MDH-3PROG-16, wateP 0.45 1.24 1.31 3.83 10.70 32.60
E,&, kcal/mol above 30 O C 33.2 a
38.20
38.20
38.20
Protein concentration equaled 5 X 10-3 mg/mL.
of these constants, as a rule, is complicated owing to the effect of the initial protein concentration on their values (18-22,36). Only an analysis of the temperature dependences of ko constants, obtained by an extrapolation of kin to %era* concentration of a biocatalyst (see Figure 71,can have a definite physical meaning. As shown in Figure 8, the Arrhenius dependence of ko has no breaks. I t should be mentioned that the thermodynamic activation parameters (AH*, AS*, and AG*),obtained from the temperature dependences of ko, cannot be related to a definite step of the protein thermoinactivation, since these pa-
rameters characterize the complex cooperative process of the conformational transformation of a protein. Ea&for the GGPDH thermoinactivation, equal to 109.7 kcal/mol, may be related to the entire stage of the dissociation of oligomeric protein into its monomers but not to a cleavage of some definite bonds which can initiate this cooperative process. The cooperative character of biopolymer therknoinactivation is a main difference between this process and thermolysis of low molecular weight conipounds. The temperature dependence of the rate constants kin reflect a nonelementary character of the thermoinactivation and a complexity of the characterized mactostage, i.e. the presence of intermediate nondisplayed microstages, the influence of the protein concentration, the solvent nature, and the enzyme microenvironment (20). Therefore, for a complete analysis of the thermodinamic and kinetic parameters of the enzyme inactivation, additional methods based on chemical kinetic theory and experimental data on much simpler systems as well as the results of the inactivation study by the methods of PMR, CD, densitometry, and sedimentational analysis should be used. CONCLUSION The catalytic activities of synthesized conjugates and their thermostabilities are determined by the DMFA content in a medium, the reaction time, and the modification degree of the dehydrogenases used. Decrease of the thermostability of dehydrogenases and their conjugates is connected with the oligomeric enzyme dissociation into subunits, which may be limiting stage of the entire process of the biocatalyst inactivation. DMFA solubilizes the dehydrogenase subunits by substituting for the structural water and, probably, initiates the enzyme dissociation into monomers. As concerns the thermostability of dehydrogenases and their conjugates, only the constants ko, obtained by an extrapolation of the effective rate constants, kin, to "zeron concentration of a biocata-
316 Bloconlrrgate Chem., Vol. 2, No. 5, 1991
lyst, are valid. The temperature dependences of the constant ko are always describedby the Anhenius equation, while those for ki, of the dehydrogenases and their conjugates are complicated by the breaks that are caused by the kin dependence on the protein concentration. Unfortunately, ko may not be calculated for all enzyme conjugates. For instance, the kin dependences on the GGPDH-3-PROG concentrations are very complex; that is accounted for the protein association owing to its hydrophobization by steroids. At small modification degrees of dehydrogenases, only the external lysine residues of a protein react with a modifier, which does not change the biocatalyst activity but decreases its stability in aqueous medium. At higher modification degrees of dehydrogenases, not only external but internal lysine residues as well react with a modifier, which, in one case, activates and stabilizes an enzyme (MDH) and, in another case, inactivates and destabilizes it (GGPDH) owing to aggregation of the hydrophobizated molecules of an enzyme. LITERATURE CITED (1) Laane, C. (1987)Medium engineering for bioorganicsynthesis. Biocatalysis I, 17-22. (2) Khmelnitski, Yu. L., Levashov, A. V., Klyachko, N. L., and Martinek, K. (1988) Engineering Biocatalytic Systems in Organic Media with Low Water Content. Enzyme Microb. Technol. 10,710-724. (3) Maggio, E. D., Ed. (1983)Enzyme-Immunoassay,CRC Press, Boca Raton, FL. (4) Ngo, T. T., and Lenhoff,H. M., Eds. (1985) Enzyme-mediated Immunoassay, Plenum Press, New York and London. (5) Means, G. E., and Feeney, R. E. (1990) Chemical Modifications of Proteins: History and Applications. Bioconjugate Chem. I, 2-12. (6) Luisi, P. L., and Magid,L. J. (1986) SolubilizationofEnzymes and Nucleic Acids in Hydrocarbon Micellar Solutions. CRC Crit. Rev. Biochem. 20, 409-474. (7) Martinek,K.,Levashov,A. V.,Klyachko,N. L.,Khmelnitaki, Yu.L., and Berezin, I. V. (1986) Micellar Enzymology Eur. J. Biochem. 155,453-468. (8) Metelitza, D. I., and Eryomin, A. N. (1988) Reversed micelles of surfactants in organic solvents as models of biomembranes (in Russian). Rev. Biol. Chem. 28, 145-173. (9) Eryomin, A. N., Savenkova, M. I., and Metelitza, D. I. (1986) The peculiarities of "antigen-antibody" interaction in the reversed micelles of surfactants in heptane (in Russian). Bioorg. Khimiya 12, 606-612. (10) Eryomin,A. N., and Metelitza,D. 1. (1989)The homogeneous enzyme-immunoassayof progesterone in the reversed micelles of surfactants (in Russian). Dokl. AN BSSR 33, 932-935. (11) Anderson, G. W., Zimmerman, J. E., and Callahan, F. M. (1964) The use of N-hydroxysuccinimidein peptide synthesis. J. Am. Chem. SOC.86, 1839-1842. Janovski, A. H. (1974) Selective 3-0-carboxymethyloxime formation in steroidal 3,20-diones for haptene immunospecificity. Steroides 23,4959. (12) Karasyova,E. I., Eryomin, A. N., and Metelitza, D. 1. (1987) Production and Characterization of the Glucose-6-phosphate DehydrogenaseConjugateswith Cortisol and Progesterone (in Russian). Biotechnology 3, 198-203. (13) Blanford, A. T.,Wittman, W., Stroup, S. D., and Westphal, U. (1978) J. Steroid Biochem. 9, 187-189. (14) Karasyova,E. I., Eryomin,A. N.,andMetelitza,D. I. (1986) Optimization of the glucose-6-phosphate dehydrogenase use in the conditionsof cofactor chemical regeneration(in Russian). IZV.AN BSSR 4,76-80. (15) Markina, V. L., Eryomin, A. N., and Metelitza, D. I. (1986) Optimum conditions of the malate and lactate dehydrogenase use at the chemical regenerationof cofactor (in Russian). Appl. Biochem. Microbiol. 22, 635-641. (16) Viola, R. E. (1984) Kinetic study of the reactions catalyzed by glucose-6-phosphatedehydrogenasefrom Leuconostoc mesenteroides: pH variation of kinetic parameters. Arch. Biochem. Biophys. 228,415-424.
Metelk8 et el.
(17) Tungler, P., and Pfleideter, G. (1977) Enhanced heat, alkalineand tryptic stability of acetamidhated pig heart lactate dehydrogenase. Biochim. Biophys. Acta 484,143. (18) Markina, V. L., Eryomin, A. N., and Metelitza, D. I. (1985) Peculiarities of the malate dehydrogenasethermoinactivation (in Russian). Biophysika 30, 971-976. (19) Markina, V. L., Eryomin, A. N., and Metelitza, D. I. (1986) Thermoinactivation and stabilization of lactate- and malatedehydrogenases (in Russian). Izo. AN BSSR 6, 61-67. (20) Metelitza, D. I., and Eryomin, A. N. (1987) Kinetic aspects of irreversible thermoinactivation of enzymes (in Russian). Usp. Khim. 56, 1921-1948. (21) Karasyova,E. I.,Eryomin, A. N., and Metelitza, D. I. (1990) Effects of the glucose-6-phosphatedehydrogenase hydrophobization by progesterone on the enzyme thermoinactivation (in Russian). Appl. Biochem. Microbiol. 26, 342-348. (22) Markina, V. L., Eryomin, A. N., and Metelitza, D. I. (1990) Influence of lactate and malate dehydrogenase modification on their stability and activity (in Russian). Biophysika 35, 30-35. (23) Sadana, A., and Henley, J. P. (1986) Effects of chemical modification on enzymatic activities and stabilities. Biotech. Bioeng. 88, 256-268. (24) Muller, J., and Klein, C. (1982) Stability of dehydrogenases 111. Malate dehydrogenase. Biochim.Biophys. Acta 707,133141. (25) Mozhaev, V. V., Martinek, K., and Berezin, I. V. (1987) Chemical,physical and biological approaches to development of stabilized enzymatic biocatalysts for biotechnology (in Russian). Usp. Khim. 56, 1659-1692. (26) Kutuzova, G. D., Ugarova, N. N., and Berizin, I. V. (1984) Total regularities of changes of the proteins and enzymes thermostability owing to chemicalmodification of their functional groups (in Russian). Usp. Khim. 53, 1852-1890. (27) Kutuzova, G. D., and Ugarova, N. N. (1986) Chemical modification as the production method for stabilized forms of the biocatalysts. In Itogi nauki i tekhniki. Biotechnologia V.5 (in Russian), pp 5-49, Viniti, Moscow. (28) Shore, J. D., and Chakraboti, S. K. (1978) Subunit dissociation of mitochondrialmalate dehydrogenase. Biochemistry 15,875-879. (29) Wood, D. C., Hodges, C. T., and Harrison, J. H. (1978) Relation of the pH and concentration-dependent dissociation of porcine heart mitochondrial malate dehydrogenase. Biochem. Biophys. Res. Commun. 82,943-950. (30) Bleibe, D. M., Schulz, R. A., and Harrison, J. H. (1977) Investigation of the subunit interaction of malate dehydrogenase. J.Biol. Chem. 252, 755-758. (31) Carson, P. E., Schrier, S. L., and Kellermeyer,R. W. (1959) Mechanism of inactivation of glucose-6-phosphate dehydrogenase in human erythrocytes. Nature 184, 1293-1295. (32) Kirkman, H. N. (1962) Glucose-6-phosphate dehydrogenase from human erythrocytes. Further purification and characterization. J. Biol. Chem. 237, 2364-2370. (33) Chang, A. E., and Langdo, E. G. (1963)J. B i d . Chem. 23092312. (34) Yoshida, A., and Hoagland, V. D. (1970) Active molecular unit and NADP content of human glucose-6-phosphate dehydrogenase. Biochem. Biophys. Res. Commun. 40, 11671172. (35) Puchkaev, A. V., and Metelitza, D. I. Catalytic activity of glucose-6-phosphate dehydrogenase in water-organic media (in Russian). Appl. Biochem. Microbiol., in press. (36) Poltorak, 0. M., and Chukhray, E. S. (1986) Dissociative thermoinactivation of biocatalysts. In Itogi nauki i tekhniki. Biotekhnologia. V.5 (in Russian), pp 50-86, Viniti, Moscow. (37) Eryomin, A. N.; Karasyova, E. I., and Metelitza, D. I. (1989) Catalytic activity of gluccee-6-phosphatedehydrogenase conjugates with progesterone in aqueous and micellar media in the presence of specificand nonspecific antibodies (in Russian). Appl. Biochem. Microbiol. 25, 524-531. (38) Shultz, G. E., and Schirmer, R. H. (1979) Principles of Protein Structure, Springer-Verlag, New York. (39) Jenks, W. P. (1969)Catalysis in Chemlstry and Enzymology (pp 303-331 in Russian translation, 1972, Mir, Moscow) McGraw-Hill, New York.
