ENZYMATIC ACTIVITY OF STAPHYLOCOAGULASE

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1 ENZYMATIC ACTIVITY OF STAPHYLOCOAGULASE I. CHARACTERIZATION OF AN ESTERASE ASSOCIATED WITH PURIFIED PREPARATIONS' MIARGARET C. DRUMMOND2 AND MORRIS TAGER Department of Bacteriology and Immunology, Emory University, Emory University, Georgia Received for publication March 9, 1959 Tager (1952, unpublished data) observed that when a purified preparation of staphylocoagulase was incubated with egg yolk, turbidity was produced similar to that formed by the toxin (lecithinase) of Clostridium perfringens. The production of opacity in egg yolk media occurs with a variety of microorganisms, such as other clostridia (Crook, 1942), aerobic sporeforming bacilli (Mc- Gaughey and Chu, 1948), and staphylococei (Gillespie and Alder, 1952). The demonstration of a second biological activity in purified staphylocoagulase made it pertinent to characterie this action in terms of substrate specificity and other general properties. This report is primarily concerned with this problem. Also it seemed important to determine whether the action on egg yolk represents an integral part of the coagulase activity, or is separable from it. The following paper will present the findings of these studies. MATERIALS AND METHODS Preparation of staphylocoagulase. Two preparations of coagulase were employed. The materials were purified from 5-day culture filtrates of Staphylococcus aureus strain 14 (Tager, 1948). Ultracentrifugation at 59,7 rpm (Spinco analytical ultracentrifuge) showed the preparations contained a single, fairly homogeneous peak with uncorrected sedimentation constants of 1.44 and 1.4. Both preparations exhibited similar plasma clotting (method of Tager and Hales, 1947) and esterase activities. After dialysis, the preparations were lyophilied I This study was supported in part by research grant no. H-1895 from the National Heart Institute of the National Institutes of Health, U. S. Public Health Service. 2 This work is in part based on a dissertation submitted in partial fulfillment of the requirements for the Ph.D. degree, Division of Basic Health Sciences, Emory University. and stored at -2 C. Dialysis was carried out for a limited time only (24 hr at 6 to 8 C) because of the lability of coagulase. This procedure yields a product which, on a dry weight basis, is 32 to 37 per cent protein (Lowry et al., 1951) and.5 per cent carbohydrate by the anthrone method (Umbreit et al., 1957). A solution of 2 mg dry weight (6.4 to 7.4 mg protein) per ml gives a clotting titer of 1:72 at 1 min, 1:144 at 2 min, and 1:112,64 at 18 hr, employing the clotting assay method described by Tager and Hales (1947). Thus 1,ug of coagulase protein results in a firm 4+ clot in 1 min, 5,ig in 2 min, and less than.1,ug in 18 hr, when coagulase and noninhibitory human plasma are mixed in equal volumes. Measurement of lecithinase activity. Lecithinase was measured by determining the rate of increase of acid-soluble phosphorus (Macfarlane and Knight, 1941). After incubation of coagulase (.5 mg per ml) and egg yolk (at a final dilution of 1:8, v/v) at 37 C for 24 hr, the reaction was terminated by the addition of 2 per cent trichloroacetic acid to give a final concentration of 5 per cent. The total phosphorus in the supernatant solution was measured by a modified Fiske and SubbaRow spectrophotometric method (Umbreit et al., 1957). Manometric assay of esterase activity. All solutions and reagents were gassed with 1 per cent carbon dioxide before use. The coagulase preparation was pipetted into the main compartment of a standard 15-ml Warburg reaction vessel along with bicarbonate and water to the total calibration volume. The substrate, emulsified in 5 per cent gum acacia and bicarbonate of a molarity corresponding to that in the main compartment, was pipetted into the side arm. This precaution is designed to prevent the initial nonspecific evolution of gas when enyme and substrate of unequal ph are mixed (Singer and Hofstee, 1948). The final molarity of bicarbonate 47

2 48 DRUMMOND AND TAGER [vol. 78 employed was 5.8 X 1-2, which yields a ph of 8., in an atmosphere of 5 per cent carbon dioxide and 95 per cent nitrogen, at 38 C. In experiments where the ph or the temperature were varied, the strength of bicarbonate was adjusted accordingly, employing the Henderson-Hasselbalch equation, and checking the final ph with a Beckman ph meter. All reaction vessels were gassed for 7 to 1 min with the carbon dioxide and nitrogen gas mixture (5:95) (per cent), and equilibrated for another 1 min in a closed system. At this time the contents of the side arm were tipped in, and the evolution of gas was observed. The stock coagulase solution was maintained under conditions of maximal stability, in 2 per cent peptone-saline, until use, when it was diluted to an appropriate concentration with distilled water. The evolution of CO2 by substrate alone and by coagulase alone was negligible under the usual conditions of assay. Carbon dioxide retention by the coagulase preparation, measured by the method of Bain (1957), was found not to be significant. RESULTS Evidence of esterase activity. Since staphylocoagulase produces a change in egg yolk stability grossly indistinguishable from the action of the a-toxin (lecithinase) of C. perfringens, preliminary attempts were made to demonstrate similar lecithinase activity in the coagulase preparation. Measurements of acid-soluble phosphorus before and after egg yolk opacity production (Macfarlane and Knight, 1941; Chu, 1949) failed to indicate such lecithinase activity in coagulase. Similarly, when purified egg yolk lecithin and lecithin derived from animal sources were employed as substrates, no release of acid-soluble phosphorus was detected. During the course of the incubation of coagulase and egg yolk in an unbuffered medium, it was noted that a significant production of acid occurred. This finding suggested the possibility of lipolytic activity. The evolution of carbon dioxide from a bicarbonate system, employing the conventional manometric method for lipase assay, further confirmed this possibility. Nature of the reaction. In the manometric assay of coagulase, tributyrin was employed at a concentration of 1 X 1-2 M and bicarbonate at 5.88 X 1-2 M. The temperature was maintained at 38 C and the resulting ph was 8.. Under these 32 Id a., 24 16, COAG CONC IN JAG PROTEIN PER ML Figure 1. Relation between coagulase concentration and the evolution of carbon dioxide at ph 8., 38 C. The substrate was 1 X 1-2 M tributyrin. conditions, there was a linear relationship between carbon dioxide evolution and time for at least 3 min. In those cases in this study where a 6-min reaction time has been chosen, this reaction time too has been found to lie within the limits of a ero-order reaction, where the rate is constant, and independent of substrate concentration. Under these conditions the rate of the reaction was found to be proportional to the enyme concentration. Figure 1 illustrates the proportionality described. Substrate specificity. In view of the original observation of a reaction between egg yolk and coagulase, the initial manometric studies were carried out employing egg yolk as substrate for the coagulase. The complexity of egg yolk, however, precludes the ready identification of the specific component which is hydrolyed by coagulase. For this reason, the relative rates of hydrolysis of synthetic esters, both glyceryl and methyl, as well as of several complex oils, were investigated. All reactions were carried out with purified coagulase at a final concentration of 2 X 1- mg per ml, at ph 8., with the various substrate materials at the following concentrations: all

3 1959] ENZYMATIC ACTIVITY OF STAPHYLOCOAGULASE. glyceryl esters at 1 X 1-2 M, methyl esters at 3 X 1-2 M, and oils at a concentration (6.5 per cent) calculated on the basis that the molecular weight was that of oleic acid. All substrate solutions were prepared in a diluent of bicarbonate and 5 per cent gum acacia, and were shaken vigorously prior to use in order to facilitate emulsification. Controls were performed simultaneously in all cases to determine the extent of the nonenymatic hydrolysis of both the substrate and of the enyme. Of the 18 substrate materials studied under the conditions of assay described, only the glyceryl esters of butyric acid, tributyrin and mono-nbutyrin, were found to be hydrolyed by purified coagulase (table 1). In view of the marked specificity of coagulase for tributyrin, this activity of the coagulase preparations has been designated a tributyrinase. It should be noted, however, that under other test conditions, namely, increased enyme concentration and prolonged reaction time, it has been possible to detect some eny- TABLE 1 Hydrolysis of various substrates by coagulase Substrate Reaction Velocity* Glyceryl esters: Triacetin... Tributyrin Trihexanoin... Trioctanoin... Trilaurin... Trimyristin... Tripalmitin... Triolein... Tristearin... Methyl esters: Methyl acetate... Methyl butyrate... Methyl caprylate. Methyl palmitate... Methyl oleate... Miscellaneous: Mono-o-butyrin Peanut oil... Olive oil... Coconut oil... Egg yolk * Reaction velocity in jal, CO2 per 6 min; ph 8., 5.8 X 1-2 M bicarbonate. Coagulase concentration for all substrates except egg yolk, 7,ug protein per ml; for egg yolk, 35,g per ml..2 IX I 1OO 1 I /s Figure 2. Lineweaver-Burk double reciprocal plot showing the effect of substrate concentration on the velocity of the coagulase-tributyrin reaction. matic activity on a substrate of cocoanut oil. The relation of the tributyrinase described to the egg yolk opacity-producing factor remains to be determined. Effect of substrate concentration on the coagulasetributyrin reaction. The hydrolysis of tributyrin by coagulase was studied manometrically at several concentrations of the substrate, ranging from 5 X 1-4 to 2 X 1-1 M. A Lineweaver-Burk double reciprocal plot of these data (figure 2) reveals inhibition of the reaction at high substrate concentrations. Similar inhibition with high concentrations of ethyl butyrate was found by Murray (193) in the case of sheep liver aliesterase. It has been suggested (Dixon and Webb, 1958) that enyme inhibition by high substrate concentrations may be due, among other causes, to the formation of an ineffective enyme-substrate complex by the crowding of substrate molecules on an enyme possessing two or more active centers. Effects of ph on the coagulase-tributyrin reaction. The coagulase-tributyrin reaction was performed at several different ph values within the limits of 6. and 8.. Because of the limitations of the manometric method, no measurements were carried out above this value. The results, shown in figure 3, indicate that the optimal ph of the catalyed reaction lies at or about ph 8.. It was necessary, however, to study separately the effects of ph on the stability of the enyme in order to determine whether or not ph 8. was the optimum for the catalyed reaction or simply for the stability of the enyme. Samples of 1 ml of the enyme were adjusted to ph 6., 6.5, 7., I 49

4 ~~~~~~~~~~~~Figure 41 DRUMMOND AND TAGER [VOL , and 8. with either.1 N HCl or NaOH. After incubation at 38 C for 6 min, the samples were neutralied by a predetermined volume of acid or alkali, diluted to an appropriate volume, 1 to W. 5 N to crw 12 a ph 5. 2 LJ I-~ 8 2 CQ -J 4 susrae CY Clsdciceoent'h uhae 15 mi;coe tinls 2 i;oentinl for 4 3;coe quie, i; pnsurs ph e.effect tepehtr OI1 hydolsito of Figurie 3. Effect of ph oni the hydrolysis of 15min the 2se3agls 4 5n 6ntragls tributyrin by coagulase, determined by the manometric method. Inset shows the effect of ph on 3minactIvaetio sofsth the stability of tributyrinase in the absence of iatoibetiirs ofrder.iexprimntase tributyrinase,i theabsenceiof conditheonseinctext substrate. Experimental conditions in text. to be first order. Experimental conditions in text. 16 and assayed at ph 8.. The results of this study i (inset to figure 3) showed the ph of maximal stability to be 7., rather than 8., the optimal ph 12 for the catalyed reaction. a Effects of temperature on the coagulase-tributyrin reaction. Temperature optimum of the a catalyed reaction. In order to establish the optimal so N temperature for the coagulase-tributyrin reaction, -J the reaction was carried out at 28, 3, 34, 38, and 4 C. Because of the dependence of buffer concentration on temperature (Umbreit et al., 1957), it C) was necessary to vary the bicarbonate concentra- w 4 tion accordingly, utiliing the Henderson-Hasselbalch equation The velocity of the reaction increased with a TEMPERATURE (c) corresponding increase in temperature, as can be Figulr-e 4. Effect of temperature on the hydrolysis of tributyrin by coagulase. at about 38 C. At 4 C the activity seen from figure 4, until a maximum was reached diminished.

5 1959] ENZYMATIC ACTIVITY OF STAPHYLOCOAGULASE. I 411 Effect of heat on enyme stability. Samples of 5. ml of the enyme were held at 5 C for intervals of time varying from 5 to 3 min. At the end of each time interval, the volume was adjusted to the original 5. ml volume, if required, and the sample placed in an ice bath until subsequent assay at 38 C. From the time course of the reaction (figure 5), it can be seen that the activity of the enyme (calculated from the final slopes in all cases) decreases progressively with the time of heating at 5 C. A plot of CO2 evolution at 6 min as a function of the time of heating at 5 C failed to yield a straight line, indicating that heat inactivation is not ero order. A plot of the logarithm of the 6-min reaction velocity versus time of heating at 5 C (inset of figure 5) suggests that the process is first order. DISCUSSION A highly purified preparation of staphylocoagulase has been observed to produce opacity in an egg yolk medium. Several microorganisms are known to affect this egg yolk opacity reaction which may in fact be accomplished by these microorganisms by means of different enyme systems. Macfarlane and Knight (1941) elucidated the egg yolk opacity reaction of C. perfringens exotoxin as a lecithinase by demonstrating an increase in acid-soluble phosphorus concomitant with the production of turbidity. Subsequent chemical analyses of the products of the reaction confirmed these findings. McGaughey and Chu (1948) found that certain sporeforming aerobic bacilli produce a visibly similar reaction but showed that the substrate was not only lecithin, but cephalin as well. MacLennon (1953) points out that several species of clostridia which do not produce true lecithinase are nevertheless capable of producing turbidity in egg yolk or serum. This reaction, he suggests, is nonspecific, and is due to a disturbance in the stability of the fat emulsion, possibly the result of proteolysis. Gillespie and Alder (1952) have described opacity in egg yolk media produced both by living cultures of Staphylococcus aureus and by ammonium sulfate precipitates of the culture filtrates. These authors concluded that the enyme responsible is a lipase, whose occurrence they found not to correspond with the production of coagulase, pigment, a-, f3-, or 5-lysin by staphylococci. No information was given relative to the clotting activity of the ammonium sulfate precipitates. Since Gillespie and Alder found 51 of 138 coagulase-positive staphylococcal cultures to be negative for the opacity factor, it did not appear likely that the lipase described by these authors was identical with the esterase activity found in purified coagulase preparations. Another apparent incongruity is the fact that the lipase described by Gillespie and Alder exhibited activity on arachis oil whereas the tributyrinase of this study failed to do so. However, the conditions of assay were not identical. The question of whether the tributyrinase and the egg yolk opacity-producing factor are identical has not been resolved by these investigations. SUMMARY A purified preparation of staphylocoagulase possesses an esterase activity with a marked specificity for tributyrin. The coagulase-tributyrin reaction has been characteried as having a ph optimum at 8., and a temperature optimum at 38 C. The reaction is ero order in the presence of excess substrate. The ph of maximal stability is 7., and heat inactivation of the enyme is first order. REFERENCES BAIN, J. A Measurement of retention of acid. In Manometric techniques, pp Edited by W. W. Umbreit, R. H. Burris, and J. F. Stauffer. Burgess Publishing Co., Minneapolis. CHU, H. P The lecithinase of Bacillus cereus and its comparison with Clostridium welchii a-toxin. J. Get. Microbiol., 3, CROOK, E. M The Nagler reaction: the breakdown of lipoprotein complexes by bacterial toxiins. Brit. J. Exptl. Pathol., 23, DIXON, M. AND WEBB, E. C Enymes. Longmans, Green and Company, London. GILLESPIE, W. A. AND ALDER, V. G Production of opacity in egg-yolk media by coagulase-positive staphylococci. J. Pathol. Bacteriol., 64, LOWRY,. H., ROSEBROUGH, N. J., FARR, A. C., AND RANDALL, R. J Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, MACFARLANE, M. G. AND KNIGHT, B. C. J. G The biochemistry of bacterial toxins.

6 412 DRUMMOND AND TAGER [VOL. 78 I. The lecithinase activity of Cl. welchii toxins. Biochem. J., 35, MAcLENNON, J. D The toxicity of clostridial enymes. Trans. N. Y. Acad. Sci., 16, MlCGAUGHEY, C. A. AND CHU, H. P The egg yolk reaction of aerobic sporing bacilli. J. Gen. Microbiol., 2, MIURRAY, D. R. P. 193 The inhibition of esterases by excess substrate. Biochem. J., 24, SINGER, T. P. AND HOFSTEE, B. H. J Studies on wheat germ lipase. I. Methods of estimation, purification and general properties of enyme. Arch. Biochem., 18, TAGER, M Concentration, partial purrification, properties and nature of staphylocoagulase. Yale J. Biol. Med., 2, TAGER, M. AND HALES, H Quantitative coagulase and toxin production by staphylococci in relation to the clinical source of the organisms. Yale J. Biol. Med., 2, UMBREIT, W. W., BURRIS, R. H., AND STAUFFER, J. F Manometric techniques. Burgess Publishing Co., Minneapolis. Downloaded from on March 31, 219 by guest