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The l -amino acid oxidases of snake venom. III. Reversible inactivation of l -amino acid oxidases
The ~-Amino Acid Oxidases of Snake Venom. III. Reversible Inactivation of L-Amino Acid Oxidases 1 E. B. Keamey 2 and Thomas P. Singer From the Department
of Biochemistry, Western Reserve University Cleveland, Ohio Received
December
School of Medicine,
22, 1950
INTRODUCTION
In a preliminary note (1) we have reported that the L-amino acid oxidase of the venoms of pit vipers and of many true vipers undergoes an inactivation in the presence of small amounts of bi- and trivalent anions. Univalent anions protect the enzyme from inactivation as do catalytic concentrations of substrates and analogs of the prosthetic group. From a study of the kinetics of the reaction it has been concluded that the inactivation is not a competitive inhibition, but that an alteration of the enzyme protein is responsible for the loss of catalytic activity. Subsequent study of the phenomenon revealed (2) that the inactivation also occurs in the absence of added anions; i.e., in distilled water alone, and that it is completely reversible under appropriate conditions. Since a study of the mechanism of this inactivation seemed to offer an insight into the factors controlling the activity of this enzyme, a systematic study was undertaken to examine the chemical changes in the enzyme protein accompanying inactivation and its reversal. The experimental findings on the inactivation in water and in the presence of multivalent anions are presented in papers III and IV and a general theory to account for these observations in paper V. However, r Supported by the National Vitamin Foundation, the Williams-Waterman Fund of the Research Corporation, and the U. S. Public Health Service. Preliminary accounts of parts of this work have been presented at the meetings of the American Society of Biological Chemists at Detroit, April, 1949, and at Atlantic City, April, 1950. * During the early phases of this work the senior author held a National Cancer Inst’itute Senior Research Fellowship. 377
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a brief statement of the hypothesis will be presented here as an aid in the interpretation of the data to follow. According to our hypothesis, the inactivation of L-amino acid oxidase is the result of a spontaneous and completely reversible change in the configuration of the native enzyme. The inactivation process may be regarded as a series of consecutive reactions, involving two or more steps, which may be represented as follows: A G EH + E- + Hf, where A is the active form of the enzyme, EH the inactive enzyme, and E- the ionized form of the latter. While the over-all process is thought not to involve extensive unfolding of the protein, since no change in solubility, molecular weight, or shape has been detected, the high energy of activation of the process implies the breakage either of one bond of considerable strength or of several relatively weak linkages, such as hydrogen bonds. The distinct dependence of the equilibrium between active and inactive enzyme on pH suggests a considerable difference in the ionization of an amino acid residue in the two forms of the protein. This change in ionization is the result of the inactivation process, since the rate of reactivation is a function of pH, while the rate of inactivation is not. EXPERIMENTAL
Since the inactivation of the enzyme occurs in water solution alone, it seemed desirable to study the characteristics of the inactivation in the absence of anions which appeared to influence the reaction, and then to determine the effects of these anions on this basic process. A solution of moccasin venom at its own pH (pH 5.5-5.7), however, is stable at 30 or 38°C. for extended periods, and only as the solution is made more alkaline by careful neutralization in the cold, does inactivation occur on subsequent incubation at 30-50°C. Since it was observed early in this work that the anionic constituents of all buffers tested either prevented (univalent anions) or enhanced the inactivation (multivalent anions in high concentrations), it was necessary to adjust the solutions of venom to the desired pH range and to rely on the considerable buffering capacity of the proteins in the venom for maintenance of pH. In order to facilitate the presentation of data, it will be necessary to cite certain experiments, in which stricter maintenance
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of pH was required, which were carried out in dilute phosphate buffers. At low concentrations (l-5 X low3 M), however, as mill be shown phosphate appears to have no effect on the inactivation or its reversal, and these data are quite comparable with the observations made on t,he behavior of the enzyme in water alone. Makrials
and Methods
All venoms used in this work were obtained from Ross Allen’s Reptile Institute, Silver Springs, Florida. The only treatment these samples received prior to shipping was removal of cell debris by brief centrifugation and drying over CaClz at approximately 0”. Unless otherwise stated the experiments to be described were performed with dried venom as the source of the enzyme, without preliminary purification. The following procedure was used for the inactivation studied. When inactivation was measured in the absence of added buffers, a weighed amount of mocassin venom was dissolved in ice-cold distilled water and cautiously neutralized by dropwise addition of 1.5 X 10e2 hi NHaOH, under stirring, in an ice bath, until the desired pH was reached. In order to avoid a pH shift on dilution, the enzyme concentration during neutralization was as close as possible to the concentration desired in the experiment. -4 sample of enzyme was immediately removed as a zero-time control, and held at 0°C. with or without the addition of Cl-, under which conditions the enzyme activity does not change. (Below lO”C., no measurable inactivation or react,ivation occurs even in several days.) The rc,st of the enzyme was then incubated under varying experimental conditions. After suitable intervals, aliquots were removed, and the remaining activity was stabilized by chilling to 0” or by adding a solution of univalent anions, usually Cl-, as tris(hydroxymethyl)aminomethane.HCI (THA-THA’HCl) buffer, and then keeping the tube at 0” until the activity could be determined. The assay could be carried out at any time after chilling the aliquot but was usually performed as soon as possible. Activity was measured in the Warburg apparatus by 02 uptake with i X 10-J M tleucine as substrate, 6 X 10e2M THATHA.HCl buffer, pH 7.2, in a total volume of 3 ml., in air, at 30 or 38”. Readings were taken at IO-min. intervals for 20-30 min., and the aliquot of enzyme was so chosen as not to exceed 250 cu. mm. OX uptake in 30 min. Under these standard assay conditions the 02 uptake was strictly proportional to enzyme activity. The limitations of this assay method and the necessary precautions have been described in the preceding paper (3). It should be added that with partly inactivated enzyme samples care was taken to insure that O2 uptake was a linear function of time, such linearity indicating that the Cl- present during the assay did not alter the previously attained level of inactivation. Per cent inactivation was calculated by comparison with the unincubated zerotime sample or with a sample incubated with excess Cl-, these two types of controls usually giving identical results. In experiments of more than 4 hr. duration a few drops of toluene were used to suppress bacterial growth. This had no effect on the enzyme.
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Time Course of the Inactivation
To determine the effect of time on the inactivation, a 0.1% solution of moccasin venom was adjusted to pH 7.2 at 0°C. and incubated at 38°C. The pH of the solution at 38” was 6.6.3 Figure 1 shows the time course of the inactivation under these conditions. There is a rapid initial inactivation which reaches a maximum in about 2 hr., after which a part of the activity gradually returns, finally reaching an equilibrium value in about 8 hr. No further change was observed for at least 12 hr. thereafter. The extent of inactivation, the position of the final equilibrium, and the shape of the curve are functions of the temperature and pH. The return of activity after a maximum had been
, 2
I 4 TIME
t I 6 8 IN HOURS
Fro. 1. Time course of the inactivation
I 10
in water at 38°C.
reached was a regular finding at 38’ and higher temperatures, and this has been correlated with a change in pH during the course of the reaction. In identical experiments, it was found that the initial pH (6.6) declined regularly 0.05-0.1 pH units during the first 2 hr. A comparison of these data with the pH curve of the inactivation (Fig. 2) 8 This fall in pH was due to the large heat of ionization of the proteins of the venom in the range of neutrality. This behavior is characteristic of proteins (4), and the different pH values measured at a series of temperatures on venom neutralized to pH 7.2 at 0” are in the expected range if histidine groups were the main buffering agents. Moccasin venom is essentially free from low-molecular-weight substances which could act as buffers, In general, all pH values in this paper refer to the pH of the incubation mixtures at the temperature of the experiments, unless otherwise noted.
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reveals that this slight fall in pH was sufficient to account for the secondary decline in per cent inactivation. In phosphate buffer, 2.5 X 1O-3 M, pH 6.0 at 38”, an inactivation curve was obtained which coincided with that in Fig. 1 up to about 2 hr. (the “maximum” in Fig. l), but at that time the per cent inactivation leveled off and remained constant,
100 -
5 60> s ao3 6-O206.0
6.2 6.4 6.6 6.8 pH at 38°C.
7.0
7.2
FIG. 2. Variation of the extent of inactivation with pH at 38”. Light circles, in 2.5 X 10m3111phosphate; shaded circles, in water. In the phosphate experiment, the timing was started by adding unneutralized moccasin venom to each tube (already at 38”) containing varying ratios of KHzPO,, and K~HPOI, to give a final concentration of 1 mg. venom/ml. Samplesfor assaywereremovedinto ice at 0,3,6,8, and 10 hr. After 3 hr. the per cent inactivation was constant. The pH’s stated were obtained with a glass electrode at 38: at zero time. At 8 hr. the pH was the same within 0.01 unit. The water samples were neutralized in ice to a series of pH values and after removal of zero-time samples for assay, aliquots were brought to 38” and timing was started. The protein concentration was 6 mg./ml. Samples were removed at frequent intervals for assay. The inhibition shown is the maximum level reached (about 2 hr.) and the pH is that measured at zero time at 38”.
showing no secondary decline. It should be emphasized that since these experiments were carried out with crude moccasin venom, the slight decrease in pH with time was not necessarily connected with changes in the L-amino acid oxidase protein per se, but may have been the consequence of other enzymatic reactions, or of a gradual absorption of COz. A similar secondary decline or hump is not observed at 30”,
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and the reason for this will be discussed in the section dealing with temperature. E:jfect of pH The influence of pH on the rate and extent of inactivation was studied both in the presence of low concentrations of phosphate buffer and without added buffers. In the latter case the venom was adjusted in the cold to varying pH values which were selected so as to give a pH range of 6-7 at 38°C. The pH values at 38” were then checked on aliquots, from time to time, in the course of t,he inactivation. The main difficulty in such experiments was the fact that even relatively concentrated solutions of moccasin venom do not have a sufficient buffer capacity to prevent the slight shift in pH (0.05-0.1 pH unit) which occurs during the first 2 hr. of incubation at 38”, and therefore a slight decrease in the degree of inactivation occurred regularly after maximum inactivation was reached, just as in Fig. 1. In 2.5 X 10e3 M phosphate, however, constant pH was maintained throughout the experiment; maximum inact’ivation was regularly reached by 2 hr., and no further change occurred for 8-12 hr. The rate of inactivation, calculated as a first-order velocity constant for a reversible system (cf. Order of Reaction), was unaffected by pH, and equilibrium was reached at the same time at all pH values. Thus the main effect of pH appears to be on the final equilibrium, not on the reaction rate. The variation of the final inactivation with pH at 38” is illustrated in Fig. 2 wherein the results obtained in dilute phosphate are compared with those obtained in water. The points in phosphate are probably more accurate, but there is in general good agreement between the two types of experiments, notwithstanding the differences in experimental conditions. At pH 7.5 the inactivation does not) exceed 95ye and at’ pH 5.8 it is already a measurable quantity. Thus the pa-inactivation curve covers about 2 pH units. This fact, in conjunction with the S-shaped nature of the curve, suggests the involvement, in the relrersible inactivation of a protein group with an ionization in the range indicated. The pH corresponding to 50% inactivation is 6.55. Efect of Temperature The effect of temperature on the rate of inactivation of a protein may be studied either by keeping the pH constant at all temperatures tested or by allowing the protein itself to act as buffer, whereby the
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pH will vary with changes in temperature according to the heats of ionization of the particular protein groups which act as buffers. The former method yields erroneous values whenever the rate of inactivation is a function of pH (5,6). Since the rate of inactivation of L-amino acid oxidase is independent of pH, either method could be used for the calculations of the energy of activation. The results obt.ained at constant pH (in dilute phosphate) and with the protein serving as the only buffer (pH varying with temperature) were identical in that the same energy of activation was obtained by either method. Only the results obtained by the second method will be presented since they also illustrate the ready reversibility of the inactivation.
FIG.
:%
Inactivation
of Gamin0
acid oxidase at various conditions in text.
temperatures.
Experimental
The experiment plotted in Fig. 3 demonstrates the influence of temperature on the inactivation. The experimental conditions were comparable with those of the experiment shown in Fig. 1. After adjustment to pH 7.2 at O”, 0.1% solutions of venom were placed in water baths and rapidly raised to the temperatures indicated. Timing was started as soon as the desired temperature was reached. The temperatures were regulated to &O.OZ”C. The solid lines in the graph represent samples wherein a constant temperature was maintained throughout the experiment; in the curves represented by dotted lines the temperature was changed at the points indicated by arrows, in order to determine the effect of this treatment on the position of the equilibrium. It is seen that at 30” the inactivation develops slowly, does not level
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off until approximately 12 hr., and no hump appears in the curve, as mentioned above. At 38” the inactivation reaches a maximum in 2 hr., after which it declines and finally tends to level off after 8 hr. The experiments at 30 and 38” were followed for 25 hr., and at the end of this period the inactivations were 77ye and 50%, respectively, showing that the level of inactivation was maintained for extended periods. At 45” the rate of inactivation is considerably greater than at 38”, but a greater fraction of the enzyme remains active at the higher temperature when the reaction has reached equilibrium. In control experiments which were identical except for the presence of 1O-2 M KCl, no inactivation occurred in any of these tubes, even in 8 hr. at 45”, a striking demonstration of the heat stability of the enzyme in the presence of anions. In other experiments it was noted that at 50”, equilibrium was reached in 20 min. or less and no further change occurred for 2 hr. At 25” the inactivation developed very slowly and no equilibrium was reached in 24 hr., while at 3” no appreciable inactivation occurred in 1 week. These experiments clearly demonstrate that the rate of inactivation increases with temperature. The position of the equilibrium, which appears to vary with temperature, is actually a reflection of the differences in pH at these temperatures. It was found that when a 0.1% solution of moccasin venom was adjusted to pH 7.2 at O”, the pH of the solution at 30” was 6.85-6.9, at 38” about 6.59, and at 45”, 6.45-6.5.3 It is noteworthy that the degree of inactivation reached at 30, 38, and 45’ agrees closely with the maximum inactivation expected for these pH values at constant temperature (cf. Fig. 2). As mentioned above, experiments designed to evaluate the influence of temperature at constant pH supported this explanation; in water or in dilute phosphate there was little difference in inactivation at 30 or 38” at the same pH, although the rates differed greatly. The slow rate of the inactivation process at 30’ explains why a hump is not observed at this temperature, since the slight pH shift occurs long before apparent equilibrium is reached, and thus the equilibrium level of inactivation is maintained. An interesting aspect of the reversibility of the process is that the equilibrium ratio of active to inactive enzyme may be readily shifted by merely changing the temperature, since this is tantamount to changing the pH of the solution. Thus, when the enzyme sample at 45” was
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placed in the 30” bath (Fig. 3, dotted line), the extent of inactivation increased at the new pH until the level characteristic of the pH corresponding to 30” was established. Conversely, heating an aliquot of the enzyme from 38 to 45” rapidly restored the activity to the 45’ level. Order of the Reaction From the foregoing sections it is apparent that the rate at which inactivation occurs is dependent on the temperature, and that the extent of inactivation reached is limited by the pH of the solution. Once reached, the maximum inactivation is maintained as long as the pH is held constant. Kinetic analysis of such a curve should then be limited to the measurements from zero-time to the point of maximum inactivation or to experiments performed in dilute phosphate buffer. Attempts to determine the kinetics of the reaction during the initial phase by the graphical method demonstrated that the inactivation does not follow either a simple first- or second-order reaction. However, a rational reaction order could be demonstrated by assuming that the maximum inactivation reached (about 6770 at 2 hr. in Fig. 1) represents the equilibrium value of the reversible system k active enzyme ti inactive k’
enzyme
and by applying the rate equations for reversible system. In a reversible system where the rate-limiting step in both the forward and reverse directions is of first order, the following relation holds: dx - = k(a - x) - k’x, dt where a denotes the concentration of active enzyme at the commencement of the experiment, x the concentration of the inactive enzyme formed, and k and k’ are the velocity constants of the forward and reverse reactions, respectively (7). A simple way of demonstrating the applicability of this formulation to the inactivation process is as follows. If we designate a - x, the concentration of active enzyme remaining at any time, as y, and the concentration of active enzyme remaining at equilibrium as ye, then by integration of the above equation it can be shown that log (y - ye)
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should be a linear function of time. Since the concentration of active enzyme is proportional to the O2 uptake in 30 min. under standard conditions in the manometric test, y is the O2 consumption per 30 min. obtained with an enzyme sample withdrawn at any time during the inactivation process, and y/eis the corresponding value obtained with an enzyme sample which has reached maximum inactivation (i.e., equilibrium). Figure 4 is a plot of log (y - ye) against time for experi2.c 1.8 1.6 G Y. & 1.4 g-] 1.2 1.c 0.E
FIQ. 4. Demonstration
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20 30 a0 TIME IN MINUTES
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60
of the reaction order of the inactivation. see text. Conditions as in Fig. 1.
For explanation
ments carried out at 38 and 44.7%. Excellent linearity was obtained also at 29.9”, but the scale of the graph does not permit inclusion of these data, since at that temperature the reaction is quite slow. The first-order constants calculated from the slopes of these curves are k = 0.938 X lOA sec.+ at 29.9’, 7.76 X 10m4sec.+ at 38’, and 24.2 X 10d4sec.-l at 44.7”. It should now be possible to describe the effect of temperature by means of the energy of activation of the process. By plotting the loga-
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Fro. 5. Variation of the rate of inactiVation with temperature. Abscissa, reciprocal absolute temperature; ordinate, log of k&order velocity constant, in sec.-‘. Experimental conditions as in Fig. 3.
rithms of the first-order velocity constants given above against the reciprocal of the absolute temperature, a linear relationship is observed (Fig. 5). From the slope of this line (-9,270), the energy of activation, E, has been calculated to be 42,500 cal./mole. E$ect of Protein Concentration
The inactivation process is independent of protein concentration, within wide limits. A typical experiment is summarized in Table I. The conclusion that protein concentration is without influence on the rate or extent of inactivation is important in the interpretation of the mechanism of the process, inasmuch as it points to an intramolecular TABLE Inactivation
I
of the Enzyme at Low and High Protein
Concentrations
The venom was neutralized to pH 7.1 at a concentration of 10.5 mg./ml. Tube A, 0.6 ml. neutralized venom and 8.4 ml. HzO; tube B, 1.4 ml. neutralized venom and 0.7 ml. HzO. For activity determination l.O-ml. aliquots of tube A and O.l-ml. aliquots of tube B were pipetted into Warburg vessels and assayed as described earlier. The 02 consumption was measured for 30 min. at 30°C. Tube
Protein concentration ma/ml.
A B
0.7 7.0
Activity zero-time
of control
fgvi$ff$ .
Inactivation
cu. nznb.
cu. mm.
per cat
167.2 161.2
80.4 80.5
52 50
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reaction and it also suggests that kinetics of the inactivation and of its reversal are the same.
process
Prevention of the Inactivation It has been pointed out that Cl- at 10e2 M or higher concentration completely prevents the inactivation. The data in Table II illustrate this point by comparing the activity of enzyme samples (neutralized to pH 7.2) after incubation for varying periods in water and in lo-* M KC1 at 45 and 50°C. In other experiments, at 10e3 M concentration TABLE II of the Inactivation by KC1 Experimental conditions as in Figs. 1 and 3. The assay was performed at 30”, and l-ml. samples of the incubation mixture, containing 1 mg. neutralized venom, were used per vessel. Prevention
Temp~chre incubation “C.
45 45 45 45 50 50 50 50
Time of incubation hr.
0
2 5 8 0 0.5 2
Ox uptake remaining after incubation In 0.01 M KC1 In water cu. nm. cu. mm.
188.2a 106.4 113.0 103.3 188.1 114.0 108.6 103.1
188.2’= 199.9 199.0 200.5 188.1 205.4 200.4 196.4
Inactivation In water In 0.1 M KC1
per cent 43 40 45 39 42 45
per cent 0 0 0 0 0 0
(1The slight apparent increase in activity in KC1 on incubation was due to the fact that during neutralization the enzyme is inactivated about 57c, but the full activity of the unneutralized venom (about 200 cu. mm.) is regained upon brief incubation with KC1 at elevated temperatures4
nearly complete protection was afforded by Cl-, whereas at lo-* M concentration some 5001, protection was observed. Since prevention of the inactivation is a property of the anion, NaCI, KCI, and chlorides of organic bases show no significant differences in this respect. Other univalent anions are about as active as Cl-, as shown in the next paper of this series. 4,4 small amount of inactivation (never more than 5-loo/,) may occur even during the most careful neutralization with 0.015 M NHnOH at 0”. This inactivation was assumed to be due to local alkalinization and it was always reversed by incubation with KC1 at 38”.
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5
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60-
1
2
3
TIME
IN HOURS
6
FIQ. 6. Protection of the enzyme by riboflavin and KCl. Curve 1,0.35 mg./ml. of neutralized venom in water; curve 2, same plus 1 X 10e4M riboflavin; curve 3, same as 1, plus 1 X 10m5hf riboflavin; curve 4, same as 1, plus 1 X 10e4 M KCl. The incubation was carried out at 38°C. The values are corrected for the inhibition by riboflavin5
On a molar basis univalent anions are less active preventers than certain substances which are tightly bound by the enzyme, presumably at the active center (S), such as riboflavin, its closely related analogs, and substrates of the enzyme. Figure 6 compares the protective efficiencies of riboflavin and KC1 on the inactivation process, carried out at 38°C.6 It is apparent that 1 X 10P4 M riboflavin protected the 5 Riboflavin itself is somewhat inhibitory to the enzyme, but this inhibition is distinctly different from the inactivation under study (8). Since the inhibition by riboflavin is an instantaneous process which proceeds in the cold, and is completely irreversible, it is permissible to subtract the inhibition of an unincubated control sample by an identical concentration of riboflavin, from the experimental figures where the sample is incubated with riboflavin at elevated temperatures. This eorrection has been applied to the points on curves 2 and 3, Fig. 6. Riboflavin, 1 X low4 M, inhibited 33% at zero-time, and after several hours the activity was still 31-330/n of the zero-time control, showing complete prevention of the inactivation. Riboflavin, 1 X 10m5M, inhibited only S’%, and this correction was applied to the points on curve 3.
390 enzyme effective From trations enzyme analogs
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completely, while this concentration of KC1 was somewhat less as a protector than 1 X 1O-6 M riboflavin. the inhibition of L-amino acid oxidase by moderate concenof a series of riboflavin analogs, their relative affinity for the has been determined (8). At low concentrations these same protect the enzyme from inactivation in the order of their
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3
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-M AZOBENZENESULFONATE x 10~ FIG. 7. The effect of azobenzene sulfonate. In the main compartment of each Warburg vessel there were, in a total volume of 2 ml., 0.7 mg. unneutralized moccasin venom, azobenzene sulfonate (adjusted to pH 7.2) at the concentration indicated, and 10-s M phosphate, pH 7.2 (squares) or 10e3M THA-THA.HCI buffer, pH 7.2 (circles). After 15 min. incubation at 38” the contents of the side arm (tileucine and additional THA buffer) were tipped in so as to give in a final volume of 3 ml. an r.-leucine concentration of 3.3 X 10-Z M, and a THA concentration of 4 X 10-e M in each vessel. Addition of the substrate prevented further inactivation. Oxygen consumption was followed for 15 min. The low substrate concentration was desirable to demonstrate the effects shown and it coincides with the optimum concentration for experiments conducted in air (3).
relative affinities for the oxidase. Those analogs which were found not to be inhibitory at 10e3M concentration (8) are also without significant protective effect at any concentration tested. While L-leucine and other substrates protect the enzyme efficiently from inactivation (l), quantitative evaluation of the protection is nearly impossible because of the changing concentration of the amino acids resulting from their concomitant oxidation. Protection of the
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enzyme can be demonstrated better with analogs of the substrate which are not oxidized, but which act as competitive inhibitors. Zeller and Maritz (9) have shown that azobenzenesulfonate and related aromatic sulfonic acids are competitive inhibitors of the oxidase of Vipera aspis venom. We have been able to confirm this observation for moccasin venom. In the presence of 3 X low3 M L-leucine, azobenzenesulfonate shows little inhibition below 1 X 10h3 M concentration, while it significantly protects the enzyme from inactivation. At higher concentrations the inhibitory power of the compound increases but so does its protective effect against the inactivation under study. This is shown in Fig. 7. In this experiment the enzyme was incubated with varying concentrations of azobenzene sulfonate in the main compartment of a Warburg vessel in the presence of phosphate (Fig. 7, squares), or in THA-THAeHCl (circles). In the latter case the Cl- in the THA buffer prevented inactivation and this series is therefore a measure of the inhibition caused by the azobenzene sulfonate alone. It is seen that while in the control series activity decreases with increasing inhibitor concentration, as expected in the phosphate series activity goes through a maximum and then declines. The shape of this curve (squares) is explained by the fact that two processes are being measured: protection from the inactivation of the enzyme and inhibition by the azobenzene sulfonate per se. Both of these increase with increasing azobenzene sulfonate concentration. The maximum in the curve is the concentration at which the protective action is greatest compared with the inhibitory effect. It is apparent, however, that the two curves asymptotically approach each other, indicating that even at the highest concentration tested prevention of the inactivation was not complete. Amino acids which are not oxidized by the enzyme (3) and do not act as competitive inhibitors are without effect on the inactivation. Among those tested which fall into this category are n-methionine, fl-alanine, nn-o-amino butyrate, in the concentration range of low2 to lop3 M, and L-proline and L-hydroxyproline, at 10V3to 10M5LV.~ Compounds other than those in the three classes of substances discussed have not been found to prevent the inactivation process. The following nonionizable compounds tested at low2 to 10-l M concenB IrProline, 1 X lOme M, inhibits the oxidase about 8% in the presence of 7 X 10-s M L-leucine. Zeller et al. have shown that the enzyme from V. aspis is competitively inhibited by bproline at very high concentrations (10).
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t&ion were without effect on the oxidase or on the inactivation ess: glucose, ribose, sorbitol, sucrose, and glycerol.
proc-
Reversal of the Inactivation Certain experiments, notably in Fig. 3, have already indicated ready reversibility of the inactivation. The present section will devoted to a study of the conditions required for reactivation of enzyme. In general, substances which prevent the inactivation promote reverse process. Reactivation is most readily demonstrated therefore
the be the the by
HOURS AT 38” FIG. 8. Time course of the reactivation by Cl-. Moccasin venom neutralized to pH 7.0 at 0” at a concentration of 0.7 mg./ml., pH about 6.6 at 38”. An initial sample was taken immediately for assay; the rest of the enzyme was warmed to 38” and incubated, and a sample was removed for assay at 1 hr. At the point indicated by the arrow, half of the incubated enzyme was mixed with an equal volume of 2 X lo+ M KCl, and the other half with an equal volume of water. The incubation at 38” was then continued and samples were periodically assayed from both tubes. The clear circles represent the further inactivation in water and the shaded circles the gradual return of activity in 1 X 10d2M KCI.
the addition of Cl- to the inactivated enzymes and continued incubation, as in Fig. 8. It is evident from this experiment that reactivation, like inactivation, is a time reaction, which takes several hours at 38” and pH 6.6 to come to completion. Since the inactivation process conforms to the characteristics of a reversible reaction wherein both the forward and reverse processes are of first order, the velocity constant of the reactivation may be calculated from the known values of the forward rate and of the equilibrium constant, since k/k’ = K,,. In agreement with this, the data in Fig. 8
REVERSIBLE
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TABLE
L-AMINO
SCID
393
OXIDASE
III
Effect of Cl- on Reactivation
Experimental conditions as in Fig. 8. Fraction of original activity per cent
02 uptake of aliquot cu. mm.
Treatment
None 2.5 hr. at 38°C. in H20 Same + 2 hr. at 38” in Hz0 pls above, but in 10m4M KC1 As above, but in 1OP M KC1 As above, but in 10e2 M ItCl
58 50 65 78 84
180 105 91 116 141 151
fit a reversible first-order reaction. However, the absolute value of the velocity constant cannot be compared with the value calculated from the equilibrium constant, since the Cl- present in this experiment displaces the equilibrium in favor of the active enzyme. The extent of reactivation, at constant pH and temperature, is also a function of the concentration of univalent anions, as seen in Table III. The rate of reactivation, like the rate of inactivation, increases with temperature. Below 38”C., reactivation is quite slow; at lo”, no reactivation occurs in 24 hr.; while at 55”, reactivation proceeds considerably faster than at 38”, as shown in Table IV. Reversal of inactivation was essentially complete in 15 min. at 55” in 1OP’ M KCl. Significant TABLE
IV at 65°C. in Water and KC1 Moccasin venom neutralized to pH 7.0 at 1.4 mg./ml. concentration at 0°C. Activity of enzyme, prior to neutralization, 182 cu. mm. in 30 min./O.5 ml. aliquot containing 0.7 mg. venom, at 38”C., under the usual assay conditions. The neutralized enzyme was incubated for 30 min. at 38”C., at 0.7 mg./ml. concentration. At this time the activity was 93 cu. mm./ml. aliquot and the inactivation 49%. Inactivation was stopped by cooling to 0”, and half of the enzyme was mixed with an equal volume of water, the other half with an equal volume of 2 X 10m2M KCl. Both tubes were rapidly warmed to 55” and the timing was started when this temperature was reached. Comparison
of the Reactivation
0s uptake cu. vnw.
lnI%z tivation per cent
Time 5&. min.
0” 5
10 15
93.6
142.4 128.9 114.2
48.6 21.8 29.2 37.3
RertCtivation per cent
-
55.1 39.9 23.3
In lO;;,fOz uptake cu. mm.
96.7
165.2 166.7 172.7
KC1
tivation per cent
46.9 9.2
8.4 5.1
RWC-
tivation per cent
-
80.4 82.1 89.6
0 The zero time refers to aliquots taken from both tubes prior to heating to 55”.
394
KEARNEY
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reactivation occurred even in the absence of added Cl-, since an increase in temperature results in a lower pH (cf. Fig. 3). The secondary decline in activity after 5 min. at 55”, in the absence of Cl-, appears to be an irreversible heat denaturation, from which KC1 protects the enzyme under the experimental conditions. At temperatures higher than 55”, heat denaturation complicates reversal studies even in the presence of 10-l M KC!l.? The effect of pH on the reactivation was tested as follows. A 1% solution of neutralized venom was inactivated at 38” to the extent of
100
1
2 3 4 TIME IN HOURS
5
FIQ. 9. Effect of pH on the reactivation. The pH values given are those of the reaction mixtures at 38” in lo-2 M imidazole buffer during reactivation. The assay was performed under the usual conditions except that 6.6 X lo+ M imidazole buffer at pH 7.2 was used and the temperature was 30”. Other conditions in text.
35%,, after which 6 aliquots of 4 ml. each were placed in separate test tubes. To these were added 1.6 ml. of 0.1 M KCl, 1.6 ml. water and, 0.8 ml. of 0.1 M imidazole buffer, to give a series of pH values between 6.3 and 7.2. Thus the imidazole concentration was 1 X lo-* M and the Cl- was 2 X 1O-2 M or higher.8 The tubes were then replaced in a 7 While KC1 does not protect the enzyme from heat denaturation very efficiently, substrates such as bleucine protect it 100% even at 73°C. (3). * The imidazole was neutralized with HCI to give the desired pH values. Thus the [Cl-] was higher at the more acid pH values. This did not alter the course of reactivation since 2 X lo* M Cl- exerts maximal effect on reactivation.
REVERSIBLE
INACTIVATION
OF L-AMINO ACID OXIDASE
395
38” bath. Samples were periodically removed from each tube for assay, and reactivation was calculated by comparison with the inactivation found just before addition of the Cl-. The results are graphically represented in Fig. 9, wherein the per cent reactivation is plotted against the incubation time with Cl-. The reactivation was not followed for a sufficiently long time to insure equilibrium at all pH values, but certain conclusions are nonetheless apparent. Thus both the extent and the rate of reversal increase as the pH decreases. This is to be expected from the fact that the equilibrium constant, which is the ratio of t,he rates of the forward and reverse reactions, is pH dependent, but the rate of inactivation (the forward reaction) is independent of pH. An unexpected feature of this experiment is the definite lag period noticeable at and above pH 6.9. The explanation of this lag is not yet at hand. ACKNOWLEDGMENTS We are greatly indebted to Dr. John T. Edsall, Harvard University; Dr. A. A. Green, Cleveland Clinic; and Dr. Linus Pauling, California Institute of Technology; for informative discussions, valuable suggestions, and for criticisms of the manuscript.
SUMMARY 1. Solutions of the L-amino acid oxidase of moccasin venom, in the crude or purified state, undergo an unusual type of spontaneous inactivation. 2. The process is entirely reversible, and the position of the apparent equilibrium between the active and inactive forms of the enzyme is determined by the pH. In the range of about pH 5.5-7.5, the higher the pH, the greater the extent of inactivation at equilibrium. 3. Temperature influences primarily the rate of the process. Below 10” no inactivation or reactivation is observed. Between 25 and 45” the rate of inactivation increases in a regular fashion, with an energy of activation of 42,500 Cal/mole. 4. Protein concentration does not seem to influence the inactivation. 5. The inactivation process follows the kinetics of a first-order reversible reaction. 6. The inactivation is completely prevented by 1O-2 M univalent anions and by low concentrations of riboflavin and those of its analogs which have a high affinity for the oxidase. Substrates and competitive inhibitors of the enzyme are also effective protective agents. At con-
396
KEARNEY
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centrations of 1O-4 to lOA M, Cl- and similar anions effect partial prevention of the inactivation. 7. Concentrations of univalent anions and of flavins which prevent the inactivation effectively, reverse it also. Reactivation, like inactivation, proceeds at a measurable rate only at elevated temperatures. In general, the higher the temperature, the more rapid is the return of activity. Reactivation is also a function of the concentration of univalent anions and of pH. Significant reversal is accomplished without added anions by merely lowering the pH. At 55” the return of activity is very rapid under these conditions. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
KEARNEY, E. B., AND SINGER, T. P., Arch. Biochem. 21, 242 (1949). KEARNEY, E. B., AND SINGER, T. P., Federation Proc. 9, 188 (1950). SINGER, T. P., AND KEARNEY, E. B., Arch. Biochem. 29, 190 (1950). COHN, E. J., AND EDSALL, J. T., Proteins, Amino Acids, and Peptides. Reinhold, New York, 1943. STEINHART, J., Kgl. Danske Videnskab. Selskab, Mat.-fys. Medd. 14, I1 (1937). LA MER, V. K., Science 86, 614 (1937). GLASSTONE, S., Textbook of Physical Chemistry. Van Nostrand, New York, 1948. SINGER, T. P., AND KEARNEY, E. B., Arch. Biochem. 27, 348 (1950). ZELLER, E. A., AND MARITZ, A., Helv. Chim. Acta 27, 1888 (1944). ZELLER, E. A., MARITZ, A., AND ISELIN, B., Helv. Chim. Acta 28, 1615 (1945).
of Biochemistry, Western Reserve University Cleveland, Ohio Received
December
School of Medicine,
22, 1950
INTRODUCTION
In a preliminary note (1) we have reported that the L-amino acid oxidase of the venoms of pit vipers and of many true vipers undergoes an inactivation in the presence of small amounts of bi- and trivalent anions. Univalent anions protect the enzyme from inactivation as do catalytic concentrations of substrates and analogs of the prosthetic group. From a study of the kinetics of the reaction it has been concluded that the inactivation is not a competitive inhibition, but that an alteration of the enzyme protein is responsible for the loss of catalytic activity. Subsequent study of the phenomenon revealed (2) that the inactivation also occurs in the absence of added anions; i.e., in distilled water alone, and that it is completely reversible under appropriate conditions. Since a study of the mechanism of this inactivation seemed to offer an insight into the factors controlling the activity of this enzyme, a systematic study was undertaken to examine the chemical changes in the enzyme protein accompanying inactivation and its reversal. The experimental findings on the inactivation in water and in the presence of multivalent anions are presented in papers III and IV and a general theory to account for these observations in paper V. However, r Supported by the National Vitamin Foundation, the Williams-Waterman Fund of the Research Corporation, and the U. S. Public Health Service. Preliminary accounts of parts of this work have been presented at the meetings of the American Society of Biological Chemists at Detroit, April, 1949, and at Atlantic City, April, 1950. * During the early phases of this work the senior author held a National Cancer Inst’itute Senior Research Fellowship. 377
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a brief statement of the hypothesis will be presented here as an aid in the interpretation of the data to follow. According to our hypothesis, the inactivation of L-amino acid oxidase is the result of a spontaneous and completely reversible change in the configuration of the native enzyme. The inactivation process may be regarded as a series of consecutive reactions, involving two or more steps, which may be represented as follows: A G EH + E- + Hf, where A is the active form of the enzyme, EH the inactive enzyme, and E- the ionized form of the latter. While the over-all process is thought not to involve extensive unfolding of the protein, since no change in solubility, molecular weight, or shape has been detected, the high energy of activation of the process implies the breakage either of one bond of considerable strength or of several relatively weak linkages, such as hydrogen bonds. The distinct dependence of the equilibrium between active and inactive enzyme on pH suggests a considerable difference in the ionization of an amino acid residue in the two forms of the protein. This change in ionization is the result of the inactivation process, since the rate of reactivation is a function of pH, while the rate of inactivation is not. EXPERIMENTAL
Since the inactivation of the enzyme occurs in water solution alone, it seemed desirable to study the characteristics of the inactivation in the absence of anions which appeared to influence the reaction, and then to determine the effects of these anions on this basic process. A solution of moccasin venom at its own pH (pH 5.5-5.7), however, is stable at 30 or 38°C. for extended periods, and only as the solution is made more alkaline by careful neutralization in the cold, does inactivation occur on subsequent incubation at 30-50°C. Since it was observed early in this work that the anionic constituents of all buffers tested either prevented (univalent anions) or enhanced the inactivation (multivalent anions in high concentrations), it was necessary to adjust the solutions of venom to the desired pH range and to rely on the considerable buffering capacity of the proteins in the venom for maintenance of pH. In order to facilitate the presentation of data, it will be necessary to cite certain experiments, in which stricter maintenance
REVERSIBLE
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OXIDrlSE
379
of pH was required, which were carried out in dilute phosphate buffers. At low concentrations (l-5 X low3 M), however, as mill be shown phosphate appears to have no effect on the inactivation or its reversal, and these data are quite comparable with the observations made on t,he behavior of the enzyme in water alone. Makrials
and Methods
All venoms used in this work were obtained from Ross Allen’s Reptile Institute, Silver Springs, Florida. The only treatment these samples received prior to shipping was removal of cell debris by brief centrifugation and drying over CaClz at approximately 0”. Unless otherwise stated the experiments to be described were performed with dried venom as the source of the enzyme, without preliminary purification. The following procedure was used for the inactivation studied. When inactivation was measured in the absence of added buffers, a weighed amount of mocassin venom was dissolved in ice-cold distilled water and cautiously neutralized by dropwise addition of 1.5 X 10e2 hi NHaOH, under stirring, in an ice bath, until the desired pH was reached. In order to avoid a pH shift on dilution, the enzyme concentration during neutralization was as close as possible to the concentration desired in the experiment. -4 sample of enzyme was immediately removed as a zero-time control, and held at 0°C. with or without the addition of Cl-, under which conditions the enzyme activity does not change. (Below lO”C., no measurable inactivation or react,ivation occurs even in several days.) The rc,st of the enzyme was then incubated under varying experimental conditions. After suitable intervals, aliquots were removed, and the remaining activity was stabilized by chilling to 0” or by adding a solution of univalent anions, usually Cl-, as tris(hydroxymethyl)aminomethane.HCI (THA-THA’HCl) buffer, and then keeping the tube at 0” until the activity could be determined. The assay could be carried out at any time after chilling the aliquot but was usually performed as soon as possible. Activity was measured in the Warburg apparatus by 02 uptake with i X 10-J M tleucine as substrate, 6 X 10e2M THATHA.HCl buffer, pH 7.2, in a total volume of 3 ml., in air, at 30 or 38”. Readings were taken at IO-min. intervals for 20-30 min., and the aliquot of enzyme was so chosen as not to exceed 250 cu. mm. OX uptake in 30 min. Under these standard assay conditions the 02 uptake was strictly proportional to enzyme activity. The limitations of this assay method and the necessary precautions have been described in the preceding paper (3). It should be added that with partly inactivated enzyme samples care was taken to insure that O2 uptake was a linear function of time, such linearity indicating that the Cl- present during the assay did not alter the previously attained level of inactivation. Per cent inactivation was calculated by comparison with the unincubated zerotime sample or with a sample incubated with excess Cl-, these two types of controls usually giving identical results. In experiments of more than 4 hr. duration a few drops of toluene were used to suppress bacterial growth. This had no effect on the enzyme.
380
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Time Course of the Inactivation
To determine the effect of time on the inactivation, a 0.1% solution of moccasin venom was adjusted to pH 7.2 at 0°C. and incubated at 38°C. The pH of the solution at 38” was 6.6.3 Figure 1 shows the time course of the inactivation under these conditions. There is a rapid initial inactivation which reaches a maximum in about 2 hr., after which a part of the activity gradually returns, finally reaching an equilibrium value in about 8 hr. No further change was observed for at least 12 hr. thereafter. The extent of inactivation, the position of the final equilibrium, and the shape of the curve are functions of the temperature and pH. The return of activity after a maximum had been
, 2
I 4 TIME
t I 6 8 IN HOURS
Fro. 1. Time course of the inactivation
I 10
in water at 38°C.
reached was a regular finding at 38’ and higher temperatures, and this has been correlated with a change in pH during the course of the reaction. In identical experiments, it was found that the initial pH (6.6) declined regularly 0.05-0.1 pH units during the first 2 hr. A comparison of these data with the pH curve of the inactivation (Fig. 2) 8 This fall in pH was due to the large heat of ionization of the proteins of the venom in the range of neutrality. This behavior is characteristic of proteins (4), and the different pH values measured at a series of temperatures on venom neutralized to pH 7.2 at 0” are in the expected range if histidine groups were the main buffering agents. Moccasin venom is essentially free from low-molecular-weight substances which could act as buffers, In general, all pH values in this paper refer to the pH of the incubation mixtures at the temperature of the experiments, unless otherwise noted.
REVERSIBLE
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OXIDASE
381
reveals that this slight fall in pH was sufficient to account for the secondary decline in per cent inactivation. In phosphate buffer, 2.5 X 1O-3 M, pH 6.0 at 38”, an inactivation curve was obtained which coincided with that in Fig. 1 up to about 2 hr. (the “maximum” in Fig. l), but at that time the per cent inactivation leveled off and remained constant,
100 -
5 60> s ao3 6-O206.0
6.2 6.4 6.6 6.8 pH at 38°C.
7.0
7.2
FIG. 2. Variation of the extent of inactivation with pH at 38”. Light circles, in 2.5 X 10m3111phosphate; shaded circles, in water. In the phosphate experiment, the timing was started by adding unneutralized moccasin venom to each tube (already at 38”) containing varying ratios of KHzPO,, and K~HPOI, to give a final concentration of 1 mg. venom/ml. Samplesfor assaywereremovedinto ice at 0,3,6,8, and 10 hr. After 3 hr. the per cent inactivation was constant. The pH’s stated were obtained with a glass electrode at 38: at zero time. At 8 hr. the pH was the same within 0.01 unit. The water samples were neutralized in ice to a series of pH values and after removal of zero-time samples for assay, aliquots were brought to 38” and timing was started. The protein concentration was 6 mg./ml. Samples were removed at frequent intervals for assay. The inhibition shown is the maximum level reached (about 2 hr.) and the pH is that measured at zero time at 38”.
showing no secondary decline. It should be emphasized that since these experiments were carried out with crude moccasin venom, the slight decrease in pH with time was not necessarily connected with changes in the L-amino acid oxidase protein per se, but may have been the consequence of other enzymatic reactions, or of a gradual absorption of COz. A similar secondary decline or hump is not observed at 30”,
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and the reason for this will be discussed in the section dealing with temperature. E:jfect of pH The influence of pH on the rate and extent of inactivation was studied both in the presence of low concentrations of phosphate buffer and without added buffers. In the latter case the venom was adjusted in the cold to varying pH values which were selected so as to give a pH range of 6-7 at 38°C. The pH values at 38” were then checked on aliquots, from time to time, in the course of t,he inactivation. The main difficulty in such experiments was the fact that even relatively concentrated solutions of moccasin venom do not have a sufficient buffer capacity to prevent the slight shift in pH (0.05-0.1 pH unit) which occurs during the first 2 hr. of incubation at 38”, and therefore a slight decrease in the degree of inactivation occurred regularly after maximum inactivation was reached, just as in Fig. 1. In 2.5 X 10e3 M phosphate, however, constant pH was maintained throughout the experiment; maximum inact’ivation was regularly reached by 2 hr., and no further change occurred for 8-12 hr. The rate of inactivation, calculated as a first-order velocity constant for a reversible system (cf. Order of Reaction), was unaffected by pH, and equilibrium was reached at the same time at all pH values. Thus the main effect of pH appears to be on the final equilibrium, not on the reaction rate. The variation of the final inactivation with pH at 38” is illustrated in Fig. 2 wherein the results obtained in dilute phosphate are compared with those obtained in water. The points in phosphate are probably more accurate, but there is in general good agreement between the two types of experiments, notwithstanding the differences in experimental conditions. At pH 7.5 the inactivation does not) exceed 95ye and at’ pH 5.8 it is already a measurable quantity. Thus the pa-inactivation curve covers about 2 pH units. This fact, in conjunction with the S-shaped nature of the curve, suggests the involvement, in the relrersible inactivation of a protein group with an ionization in the range indicated. The pH corresponding to 50% inactivation is 6.55. Efect of Temperature The effect of temperature on the rate of inactivation of a protein may be studied either by keeping the pH constant at all temperatures tested or by allowing the protein itself to act as buffer, whereby the
REVERSIBLE
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383
pH will vary with changes in temperature according to the heats of ionization of the particular protein groups which act as buffers. The former method yields erroneous values whenever the rate of inactivation is a function of pH (5,6). Since the rate of inactivation of L-amino acid oxidase is independent of pH, either method could be used for the calculations of the energy of activation. The results obt.ained at constant pH (in dilute phosphate) and with the protein serving as the only buffer (pH varying with temperature) were identical in that the same energy of activation was obtained by either method. Only the results obtained by the second method will be presented since they also illustrate the ready reversibility of the inactivation.
FIG.
:%
Inactivation
of Gamin0
acid oxidase at various conditions in text.
temperatures.
Experimental
The experiment plotted in Fig. 3 demonstrates the influence of temperature on the inactivation. The experimental conditions were comparable with those of the experiment shown in Fig. 1. After adjustment to pH 7.2 at O”, 0.1% solutions of venom were placed in water baths and rapidly raised to the temperatures indicated. Timing was started as soon as the desired temperature was reached. The temperatures were regulated to &O.OZ”C. The solid lines in the graph represent samples wherein a constant temperature was maintained throughout the experiment; in the curves represented by dotted lines the temperature was changed at the points indicated by arrows, in order to determine the effect of this treatment on the position of the equilibrium. It is seen that at 30” the inactivation develops slowly, does not level
384
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off until approximately 12 hr., and no hump appears in the curve, as mentioned above. At 38” the inactivation reaches a maximum in 2 hr., after which it declines and finally tends to level off after 8 hr. The experiments at 30 and 38” were followed for 25 hr., and at the end of this period the inactivations were 77ye and 50%, respectively, showing that the level of inactivation was maintained for extended periods. At 45” the rate of inactivation is considerably greater than at 38”, but a greater fraction of the enzyme remains active at the higher temperature when the reaction has reached equilibrium. In control experiments which were identical except for the presence of 1O-2 M KCl, no inactivation occurred in any of these tubes, even in 8 hr. at 45”, a striking demonstration of the heat stability of the enzyme in the presence of anions. In other experiments it was noted that at 50”, equilibrium was reached in 20 min. or less and no further change occurred for 2 hr. At 25” the inactivation developed very slowly and no equilibrium was reached in 24 hr., while at 3” no appreciable inactivation occurred in 1 week. These experiments clearly demonstrate that the rate of inactivation increases with temperature. The position of the equilibrium, which appears to vary with temperature, is actually a reflection of the differences in pH at these temperatures. It was found that when a 0.1% solution of moccasin venom was adjusted to pH 7.2 at O”, the pH of the solution at 30” was 6.85-6.9, at 38” about 6.59, and at 45”, 6.45-6.5.3 It is noteworthy that the degree of inactivation reached at 30, 38, and 45’ agrees closely with the maximum inactivation expected for these pH values at constant temperature (cf. Fig. 2). As mentioned above, experiments designed to evaluate the influence of temperature at constant pH supported this explanation; in water or in dilute phosphate there was little difference in inactivation at 30 or 38” at the same pH, although the rates differed greatly. The slow rate of the inactivation process at 30’ explains why a hump is not observed at this temperature, since the slight pH shift occurs long before apparent equilibrium is reached, and thus the equilibrium level of inactivation is maintained. An interesting aspect of the reversibility of the process is that the equilibrium ratio of active to inactive enzyme may be readily shifted by merely changing the temperature, since this is tantamount to changing the pH of the solution. Thus, when the enzyme sample at 45” was
REVERSIBLE
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385
placed in the 30” bath (Fig. 3, dotted line), the extent of inactivation increased at the new pH until the level characteristic of the pH corresponding to 30” was established. Conversely, heating an aliquot of the enzyme from 38 to 45” rapidly restored the activity to the 45’ level. Order of the Reaction From the foregoing sections it is apparent that the rate at which inactivation occurs is dependent on the temperature, and that the extent of inactivation reached is limited by the pH of the solution. Once reached, the maximum inactivation is maintained as long as the pH is held constant. Kinetic analysis of such a curve should then be limited to the measurements from zero-time to the point of maximum inactivation or to experiments performed in dilute phosphate buffer. Attempts to determine the kinetics of the reaction during the initial phase by the graphical method demonstrated that the inactivation does not follow either a simple first- or second-order reaction. However, a rational reaction order could be demonstrated by assuming that the maximum inactivation reached (about 6770 at 2 hr. in Fig. 1) represents the equilibrium value of the reversible system k active enzyme ti inactive k’
enzyme
and by applying the rate equations for reversible system. In a reversible system where the rate-limiting step in both the forward and reverse directions is of first order, the following relation holds: dx - = k(a - x) - k’x, dt where a denotes the concentration of active enzyme at the commencement of the experiment, x the concentration of the inactive enzyme formed, and k and k’ are the velocity constants of the forward and reverse reactions, respectively (7). A simple way of demonstrating the applicability of this formulation to the inactivation process is as follows. If we designate a - x, the concentration of active enzyme remaining at any time, as y, and the concentration of active enzyme remaining at equilibrium as ye, then by integration of the above equation it can be shown that log (y - ye)
386
KEARNEY
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should be a linear function of time. Since the concentration of active enzyme is proportional to the O2 uptake in 30 min. under standard conditions in the manometric test, y is the O2 consumption per 30 min. obtained with an enzyme sample withdrawn at any time during the inactivation process, and y/eis the corresponding value obtained with an enzyme sample which has reached maximum inactivation (i.e., equilibrium). Figure 4 is a plot of log (y - ye) against time for experi2.c 1.8 1.6 G Y. & 1.4 g-] 1.2 1.c 0.E
FIQ. 4. Demonstration
I
I
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10
20 30 a0 TIME IN MINUTES
I
I
50
60
of the reaction order of the inactivation. see text. Conditions as in Fig. 1.
For explanation
ments carried out at 38 and 44.7%. Excellent linearity was obtained also at 29.9”, but the scale of the graph does not permit inclusion of these data, since at that temperature the reaction is quite slow. The first-order constants calculated from the slopes of these curves are k = 0.938 X lOA sec.+ at 29.9’, 7.76 X 10m4sec.+ at 38’, and 24.2 X 10d4sec.-l at 44.7”. It should now be possible to describe the effect of temperature by means of the energy of activation of the process. By plotting the loga-
REVERSIBLE
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387
Fro. 5. Variation of the rate of inactiVation with temperature. Abscissa, reciprocal absolute temperature; ordinate, log of k&order velocity constant, in sec.-‘. Experimental conditions as in Fig. 3.
rithms of the first-order velocity constants given above against the reciprocal of the absolute temperature, a linear relationship is observed (Fig. 5). From the slope of this line (-9,270), the energy of activation, E, has been calculated to be 42,500 cal./mole. E$ect of Protein Concentration
The inactivation process is independent of protein concentration, within wide limits. A typical experiment is summarized in Table I. The conclusion that protein concentration is without influence on the rate or extent of inactivation is important in the interpretation of the mechanism of the process, inasmuch as it points to an intramolecular TABLE Inactivation
I
of the Enzyme at Low and High Protein
Concentrations
The venom was neutralized to pH 7.1 at a concentration of 10.5 mg./ml. Tube A, 0.6 ml. neutralized venom and 8.4 ml. HzO; tube B, 1.4 ml. neutralized venom and 0.7 ml. HzO. For activity determination l.O-ml. aliquots of tube A and O.l-ml. aliquots of tube B were pipetted into Warburg vessels and assayed as described earlier. The 02 consumption was measured for 30 min. at 30°C. Tube
Protein concentration ma/ml.
A B
0.7 7.0
Activity zero-time
of control
fgvi$ff$ .
Inactivation
cu. nznb.
cu. mm.
per cat
167.2 161.2
80.4 80.5
52 50
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reaction and it also suggests that kinetics of the inactivation and of its reversal are the same.
process
Prevention of the Inactivation It has been pointed out that Cl- at 10e2 M or higher concentration completely prevents the inactivation. The data in Table II illustrate this point by comparing the activity of enzyme samples (neutralized to pH 7.2) after incubation for varying periods in water and in lo-* M KC1 at 45 and 50°C. In other experiments, at 10e3 M concentration TABLE II of the Inactivation by KC1 Experimental conditions as in Figs. 1 and 3. The assay was performed at 30”, and l-ml. samples of the incubation mixture, containing 1 mg. neutralized venom, were used per vessel. Prevention
Temp~chre incubation “C.
45 45 45 45 50 50 50 50
Time of incubation hr.
0
2 5 8 0 0.5 2
Ox uptake remaining after incubation In 0.01 M KC1 In water cu. nm. cu. mm.
188.2a 106.4 113.0 103.3 188.1 114.0 108.6 103.1
188.2’= 199.9 199.0 200.5 188.1 205.4 200.4 196.4
Inactivation In water In 0.1 M KC1
per cent 43 40 45 39 42 45
per cent 0 0 0 0 0 0
(1The slight apparent increase in activity in KC1 on incubation was due to the fact that during neutralization the enzyme is inactivated about 57c, but the full activity of the unneutralized venom (about 200 cu. mm.) is regained upon brief incubation with KC1 at elevated temperatures4
nearly complete protection was afforded by Cl-, whereas at lo-* M concentration some 5001, protection was observed. Since prevention of the inactivation is a property of the anion, NaCI, KCI, and chlorides of organic bases show no significant differences in this respect. Other univalent anions are about as active as Cl-, as shown in the next paper of this series. 4,4 small amount of inactivation (never more than 5-loo/,) may occur even during the most careful neutralization with 0.015 M NHnOH at 0”. This inactivation was assumed to be due to local alkalinization and it was always reversed by incubation with KC1 at 38”.
REVERSIBLE
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4
5
OXIDASE
389
60-
1
2
3
TIME
IN HOURS
6
FIQ. 6. Protection of the enzyme by riboflavin and KCl. Curve 1,0.35 mg./ml. of neutralized venom in water; curve 2, same plus 1 X 10e4M riboflavin; curve 3, same as 1, plus 1 X 10m5hf riboflavin; curve 4, same as 1, plus 1 X 10e4 M KCl. The incubation was carried out at 38°C. The values are corrected for the inhibition by riboflavin5
On a molar basis univalent anions are less active preventers than certain substances which are tightly bound by the enzyme, presumably at the active center (S), such as riboflavin, its closely related analogs, and substrates of the enzyme. Figure 6 compares the protective efficiencies of riboflavin and KC1 on the inactivation process, carried out at 38°C.6 It is apparent that 1 X 10P4 M riboflavin protected the 5 Riboflavin itself is somewhat inhibitory to the enzyme, but this inhibition is distinctly different from the inactivation under study (8). Since the inhibition by riboflavin is an instantaneous process which proceeds in the cold, and is completely irreversible, it is permissible to subtract the inhibition of an unincubated control sample by an identical concentration of riboflavin, from the experimental figures where the sample is incubated with riboflavin at elevated temperatures. This eorrection has been applied to the points on curves 2 and 3, Fig. 6. Riboflavin, 1 X low4 M, inhibited 33% at zero-time, and after several hours the activity was still 31-330/n of the zero-time control, showing complete prevention of the inactivation. Riboflavin, 1 X 10m5M, inhibited only S’%, and this correction was applied to the points on curve 3.
390 enzyme effective From trations enzyme analogs
KEARNEY
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completely, while this concentration of KC1 was somewhat less as a protector than 1 X 1O-6 M riboflavin. the inhibition of L-amino acid oxidase by moderate concenof a series of riboflavin analogs, their relative affinity for the has been determined (8). At low concentrations these same protect the enzyme from inactivation in the order of their
8060
201
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1
2
3
4
5
-M AZOBENZENESULFONATE x 10~ FIG. 7. The effect of azobenzene sulfonate. In the main compartment of each Warburg vessel there were, in a total volume of 2 ml., 0.7 mg. unneutralized moccasin venom, azobenzene sulfonate (adjusted to pH 7.2) at the concentration indicated, and 10-s M phosphate, pH 7.2 (squares) or 10e3M THA-THA.HCI buffer, pH 7.2 (circles). After 15 min. incubation at 38” the contents of the side arm (tileucine and additional THA buffer) were tipped in so as to give in a final volume of 3 ml. an r.-leucine concentration of 3.3 X 10-Z M, and a THA concentration of 4 X 10-e M in each vessel. Addition of the substrate prevented further inactivation. Oxygen consumption was followed for 15 min. The low substrate concentration was desirable to demonstrate the effects shown and it coincides with the optimum concentration for experiments conducted in air (3).
relative affinities for the oxidase. Those analogs which were found not to be inhibitory at 10e3M concentration (8) are also without significant protective effect at any concentration tested. While L-leucine and other substrates protect the enzyme efficiently from inactivation (l), quantitative evaluation of the protection is nearly impossible because of the changing concentration of the amino acids resulting from their concomitant oxidation. Protection of the
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OXIDASE
391
enzyme can be demonstrated better with analogs of the substrate which are not oxidized, but which act as competitive inhibitors. Zeller and Maritz (9) have shown that azobenzenesulfonate and related aromatic sulfonic acids are competitive inhibitors of the oxidase of Vipera aspis venom. We have been able to confirm this observation for moccasin venom. In the presence of 3 X low3 M L-leucine, azobenzenesulfonate shows little inhibition below 1 X 10h3 M concentration, while it significantly protects the enzyme from inactivation. At higher concentrations the inhibitory power of the compound increases but so does its protective effect against the inactivation under study. This is shown in Fig. 7. In this experiment the enzyme was incubated with varying concentrations of azobenzene sulfonate in the main compartment of a Warburg vessel in the presence of phosphate (Fig. 7, squares), or in THA-THAeHCl (circles). In the latter case the Cl- in the THA buffer prevented inactivation and this series is therefore a measure of the inhibition caused by the azobenzene sulfonate alone. It is seen that while in the control series activity decreases with increasing inhibitor concentration, as expected in the phosphate series activity goes through a maximum and then declines. The shape of this curve (squares) is explained by the fact that two processes are being measured: protection from the inactivation of the enzyme and inhibition by the azobenzene sulfonate per se. Both of these increase with increasing azobenzene sulfonate concentration. The maximum in the curve is the concentration at which the protective action is greatest compared with the inhibitory effect. It is apparent, however, that the two curves asymptotically approach each other, indicating that even at the highest concentration tested prevention of the inactivation was not complete. Amino acids which are not oxidized by the enzyme (3) and do not act as competitive inhibitors are without effect on the inactivation. Among those tested which fall into this category are n-methionine, fl-alanine, nn-o-amino butyrate, in the concentration range of low2 to lop3 M, and L-proline and L-hydroxyproline, at 10V3to 10M5LV.~ Compounds other than those in the three classes of substances discussed have not been found to prevent the inactivation process. The following nonionizable compounds tested at low2 to 10-l M concenB IrProline, 1 X lOme M, inhibits the oxidase about 8% in the presence of 7 X 10-s M L-leucine. Zeller et al. have shown that the enzyme from V. aspis is competitively inhibited by bproline at very high concentrations (10).
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KEARNEY
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t&ion were without effect on the oxidase or on the inactivation ess: glucose, ribose, sorbitol, sucrose, and glycerol.
proc-
Reversal of the Inactivation Certain experiments, notably in Fig. 3, have already indicated ready reversibility of the inactivation. The present section will devoted to a study of the conditions required for reactivation of enzyme. In general, substances which prevent the inactivation promote reverse process. Reactivation is most readily demonstrated therefore
the be the the by
HOURS AT 38” FIG. 8. Time course of the reactivation by Cl-. Moccasin venom neutralized to pH 7.0 at 0” at a concentration of 0.7 mg./ml., pH about 6.6 at 38”. An initial sample was taken immediately for assay; the rest of the enzyme was warmed to 38” and incubated, and a sample was removed for assay at 1 hr. At the point indicated by the arrow, half of the incubated enzyme was mixed with an equal volume of 2 X lo+ M KCl, and the other half with an equal volume of water. The incubation at 38” was then continued and samples were periodically assayed from both tubes. The clear circles represent the further inactivation in water and the shaded circles the gradual return of activity in 1 X 10d2M KCI.
the addition of Cl- to the inactivated enzymes and continued incubation, as in Fig. 8. It is evident from this experiment that reactivation, like inactivation, is a time reaction, which takes several hours at 38” and pH 6.6 to come to completion. Since the inactivation process conforms to the characteristics of a reversible reaction wherein both the forward and reverse processes are of first order, the velocity constant of the reactivation may be calculated from the known values of the forward rate and of the equilibrium constant, since k/k’ = K,,. In agreement with this, the data in Fig. 8
REVERSIBLE
IiYACTIVATIOh-
OF
TABLE
L-AMINO
SCID
393
OXIDASE
III
Effect of Cl- on Reactivation
Experimental conditions as in Fig. 8. Fraction of original activity per cent
02 uptake of aliquot cu. mm.
Treatment
None 2.5 hr. at 38°C. in H20 Same + 2 hr. at 38” in Hz0 pls above, but in 10m4M KC1 As above, but in 1OP M KC1 As above, but in 10e2 M ItCl
58 50 65 78 84
180 105 91 116 141 151
fit a reversible first-order reaction. However, the absolute value of the velocity constant cannot be compared with the value calculated from the equilibrium constant, since the Cl- present in this experiment displaces the equilibrium in favor of the active enzyme. The extent of reactivation, at constant pH and temperature, is also a function of the concentration of univalent anions, as seen in Table III. The rate of reactivation, like the rate of inactivation, increases with temperature. Below 38”C., reactivation is quite slow; at lo”, no reactivation occurs in 24 hr.; while at 55”, reactivation proceeds considerably faster than at 38”, as shown in Table IV. Reversal of inactivation was essentially complete in 15 min. at 55” in 1OP’ M KCl. Significant TABLE
IV at 65°C. in Water and KC1 Moccasin venom neutralized to pH 7.0 at 1.4 mg./ml. concentration at 0°C. Activity of enzyme, prior to neutralization, 182 cu. mm. in 30 min./O.5 ml. aliquot containing 0.7 mg. venom, at 38”C., under the usual assay conditions. The neutralized enzyme was incubated for 30 min. at 38”C., at 0.7 mg./ml. concentration. At this time the activity was 93 cu. mm./ml. aliquot and the inactivation 49%. Inactivation was stopped by cooling to 0”, and half of the enzyme was mixed with an equal volume of water, the other half with an equal volume of 2 X 10m2M KCl. Both tubes were rapidly warmed to 55” and the timing was started when this temperature was reached. Comparison
of the Reactivation
0s uptake cu. vnw.
lnI%z tivation per cent
Time 5&. min.
0” 5
10 15
93.6
142.4 128.9 114.2
48.6 21.8 29.2 37.3
RertCtivation per cent
-
55.1 39.9 23.3
In lO;;,fOz uptake cu. mm.
96.7
165.2 166.7 172.7
KC1
tivation per cent
46.9 9.2
8.4 5.1
RWC-
tivation per cent
-
80.4 82.1 89.6
0 The zero time refers to aliquots taken from both tubes prior to heating to 55”.
394
KEARNEY
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reactivation occurred even in the absence of added Cl-, since an increase in temperature results in a lower pH (cf. Fig. 3). The secondary decline in activity after 5 min. at 55”, in the absence of Cl-, appears to be an irreversible heat denaturation, from which KC1 protects the enzyme under the experimental conditions. At temperatures higher than 55”, heat denaturation complicates reversal studies even in the presence of 10-l M KC!l.? The effect of pH on the reactivation was tested as follows. A 1% solution of neutralized venom was inactivated at 38” to the extent of
100
1
2 3 4 TIME IN HOURS
5
FIQ. 9. Effect of pH on the reactivation. The pH values given are those of the reaction mixtures at 38” in lo-2 M imidazole buffer during reactivation. The assay was performed under the usual conditions except that 6.6 X lo+ M imidazole buffer at pH 7.2 was used and the temperature was 30”. Other conditions in text.
35%,, after which 6 aliquots of 4 ml. each were placed in separate test tubes. To these were added 1.6 ml. of 0.1 M KCl, 1.6 ml. water and, 0.8 ml. of 0.1 M imidazole buffer, to give a series of pH values between 6.3 and 7.2. Thus the imidazole concentration was 1 X lo-* M and the Cl- was 2 X 1O-2 M or higher.8 The tubes were then replaced in a 7 While KC1 does not protect the enzyme from heat denaturation very efficiently, substrates such as bleucine protect it 100% even at 73°C. (3). * The imidazole was neutralized with HCI to give the desired pH values. Thus the [Cl-] was higher at the more acid pH values. This did not alter the course of reactivation since 2 X lo* M Cl- exerts maximal effect on reactivation.
REVERSIBLE
INACTIVATION
OF L-AMINO ACID OXIDASE
395
38” bath. Samples were periodically removed from each tube for assay, and reactivation was calculated by comparison with the inactivation found just before addition of the Cl-. The results are graphically represented in Fig. 9, wherein the per cent reactivation is plotted against the incubation time with Cl-. The reactivation was not followed for a sufficiently long time to insure equilibrium at all pH values, but certain conclusions are nonetheless apparent. Thus both the extent and the rate of reversal increase as the pH decreases. This is to be expected from the fact that the equilibrium constant, which is the ratio of t,he rates of the forward and reverse reactions, is pH dependent, but the rate of inactivation (the forward reaction) is independent of pH. An unexpected feature of this experiment is the definite lag period noticeable at and above pH 6.9. The explanation of this lag is not yet at hand. ACKNOWLEDGMENTS We are greatly indebted to Dr. John T. Edsall, Harvard University; Dr. A. A. Green, Cleveland Clinic; and Dr. Linus Pauling, California Institute of Technology; for informative discussions, valuable suggestions, and for criticisms of the manuscript.
SUMMARY 1. Solutions of the L-amino acid oxidase of moccasin venom, in the crude or purified state, undergo an unusual type of spontaneous inactivation. 2. The process is entirely reversible, and the position of the apparent equilibrium between the active and inactive forms of the enzyme is determined by the pH. In the range of about pH 5.5-7.5, the higher the pH, the greater the extent of inactivation at equilibrium. 3. Temperature influences primarily the rate of the process. Below 10” no inactivation or reactivation is observed. Between 25 and 45” the rate of inactivation increases in a regular fashion, with an energy of activation of 42,500 Cal/mole. 4. Protein concentration does not seem to influence the inactivation. 5. The inactivation process follows the kinetics of a first-order reversible reaction. 6. The inactivation is completely prevented by 1O-2 M univalent anions and by low concentrations of riboflavin and those of its analogs which have a high affinity for the oxidase. Substrates and competitive inhibitors of the enzyme are also effective protective agents. At con-
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centrations of 1O-4 to lOA M, Cl- and similar anions effect partial prevention of the inactivation. 7. Concentrations of univalent anions and of flavins which prevent the inactivation effectively, reverse it also. Reactivation, like inactivation, proceeds at a measurable rate only at elevated temperatures. In general, the higher the temperature, the more rapid is the return of activity. Reactivation is also a function of the concentration of univalent anions and of pH. Significant reversal is accomplished without added anions by merely lowering the pH. At 55” the return of activity is very rapid under these conditions. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
KEARNEY, E. B., AND SINGER, T. P., Arch. Biochem. 21, 242 (1949). KEARNEY, E. B., AND SINGER, T. P., Federation Proc. 9, 188 (1950). SINGER, T. P., AND KEARNEY, E. B., Arch. Biochem. 29, 190 (1950). COHN, E. J., AND EDSALL, J. T., Proteins, Amino Acids, and Peptides. Reinhold, New York, 1943. STEINHART, J., Kgl. Danske Videnskab. Selskab, Mat.-fys. Medd. 14, I1 (1937). LA MER, V. K., Science 86, 614 (1937). GLASSTONE, S., Textbook of Physical Chemistry. Van Nostrand, New York, 1948. SINGER, T. P., AND KEARNEY, E. B., Arch. Biochem. 27, 348 (1950). ZELLER, E. A., AND MARITZ, A., Helv. Chim. Acta 27, 1888 (1944). ZELLER, E. A., MARITZ, A., AND ISELIN, B., Helv. Chim. Acta 28, 1615 (1945).