EIGCHEMICAE. STUDIES ON STAPHYLOCOAGUMSI WITH EMPHASIS UPON M ASSOCIATED PHOSPHATAS? ACTIVITY Thesis For fI-m Degree 04’ Ph. D. MICHIGAN STATE UNIVERSITY WEIIIam E. Innis: 19M THESIS C ”1— LIB R A. R "in" 3‘ Michigan ‘3: it: ““ 'lJHWVErifiyf .me-w--- ' ""” w This is to certify that the thesis entitled Biochemical Studies on Staphylocoagulase with Emphasis upon an Associated Phosphatase Activity presented by William E. Inniss has been accepted towards fulfillment of the requirements for Ph.D. degree in Department of Microbiology 5 Public Health éfém Major professor 0-169 q. Afico‘flf’ Hi .1 , I“! U; .3yhuuu I 1n:":’: ">1 I’;..:O~IQH U" It has ’: In. ‘ .' Y. EI.:.S be see“ fl‘pI‘de ‘ but"; .LCCCCC-. "5 Isiah!“ ‘ 'V ‘vabbfi‘i 4 heart infusio "A! In 0rd rs“ N I?) \J C? 'fi‘r n “An ‘LwSeSQe-l “ A 9!; ABSTRACT BIOCHEMICAL STUDIES ON STAPHYLOCOAGULASE WITH EMPHASIS UPON AN ASSOCIATED PHOSPHATASE ACTIVITY by William E. Inniss It has been previously suggested that a correlation exists between the coagulase and phosphatase activity of staphylococci. The present investigation was carried out to determine if this relationship was a functional one. A known high coagulase producer, Staphylococcus aureus, phage propagating strain 70, was grown in brain heart infusion and the coagulase purified. The coagulase preparation was found to possess a concomitant phOSphatase activity as measured spectrophotometrically at 400 my using para-nitrophenylphosphate as the substrate. In order to clarify this dual phenomenon, a series of studies were conducted such as: thermal inactivation, anion exchange chromatography, starch, starch gel, and paper electrOphoresis, effect of inhibitors, saturation of enzyme sites, and parallel measurement of specific activities. These approaches were intended to determine (1) whether the same active group on a common protein accounted for both activities; (2) whether different active groups were associated on the same protein entity; (3) or whether two proteins, intimately associated, each possessed a different active group. Thermal inactivation experimentation, carried out on issue In N 3:212:12: to an graiiem el‘xi: 1.25 I. SaCl. :1‘ the two ac: 'ere asscciate i252 subiecte: ;::res‘.s uzde fl" 0‘“ “*‘erent 98:5 cf ‘AJK “\‘V‘~€ PA. A Q ‘~:1 ‘ 04; L William E. Inniss the purified coagulase preparation showed a parallel decrease in both activities. The preparation was subjected to anion exchange chromatography employing gradient elution of DEAE-cellulose at pH 7.5 and 8.6 with 1.25 M NaCl. The occurence of coincident maximum peaks of the two activities suggested that both functional groups were associated on the same protein. The two activities, when subjected to starch, starch gel, and paper electro- phoresis under various conditions of buffers and pH (pH 4.2 to 10), exhibited similar maximum peaks of activity after migration. An extremely small degree of separation may have been obtained when electrOphoresis was conducted with a discontinuous buffer system. However, it was not possible to recover either activity in the\ complete absence of the other because of the large amount of overlapping of the two activities. Inhibitory compounds such as EDTA, iodoacetate, fluoride, azide and para-chloromercuribenzoate exerted different rather than similar degrees of inhibition on each of the two activities indicating that the two activities do not operate by the same mechanism. This supposition was verified by subsequent experiments in which the phOSphatase was completely saturated by an excess of para-nitrophenylphosphate substrate (loo-fold K3 value). Under these conditions the coagulase activity remained intact. William E. Inniss Parallel increases were found in the total activity (on the basis of protein) for both the coagulase and phosphatase after each purification step. It is therefore hypothesized that the coagulase and phosphatase activities represent two different functional groups associated with the same or an extremely similar protein entity. BIOCHEMICAL STUDIES ON STAPHYLOCOAGULASE WITH EMPHASIS UPON AN ASSOCIATED PHOSPHATASE ACTIVITY By of William Efalnniss A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1951 0—... _m1..«r——— H L]. the a‘ In. It n : t3 arr! U. L. "" $ 0 .. 11 ucbic C v I. I" 5" *1. 11 .-r in. CJvJ he wilaV 0 G' r,- s" .Me n“. 1321*. R. 1:. ‘..,. 5.... b A: 0:..‘Aru-CZU U- 0 y ‘ ‘ P Q '3' 30 :‘.rie ACKNOWLEDGEMENTS The author wishes to express his sincere appreciation to Dr. C. L. San Clemente of the Department of Microbiology and Public Health for his interest, guidance and technical advice. The numerous helpful suggestions generously given by Dr. R. N. Costilow and Dr. H. L. Sadoff of the Department of Microbiology and Public Health and Dr. J. L. Fairley of the Department of Biochemistry were also greatly appreciated. 11 ".9. 1“" A" 7 n . mafia-w" ' ' ' .M’T.' fl? T 1H3? ‘ .24...“ V. “' 0C} H‘ ‘ CC 5 uapz'll A lime of n .. Veata Vi'?i§’!”‘ n" "‘“Ntiho A“ .. .3. . ._A..-._. A «fig-n...» TABLE OF CONTENTS PAGE INTRODUCTION....................................... 1 REVIEW OF LITERATURE............................... 3 Staphylococci and Coagulase................... 3 Mode of Action of Staphylocoagulase in the Coagulation of Plasma..................... 8 Physical and Chemical Properties of Coagulase. 12 Relationship of Coagulase to other Biological Activities of Coagulase-positive Staphylococci............................. 15 Isolation, Concentration and Purification of Coagulase................................. .19 MATERIALS AND METHODS.................{............ 23 Organisms and Culture Media.................... 23 Assay Methods.................................. 23 Coagulase................................. 23 Phosphatase............................... 24 Protein................................... 25 Isolation and Purification of Coagulase........ 26 Anion Exchange Chromatography.................. 28 ElectrOphoresis................................ 28 Starch block electrophoresis.............. 28 Starch gel electrophoresis................ 30 Paper electrophoresis..................... 31 RESULTS............................................ 32 Coagulase Production in a Biphasic and Non- iii #n—au-‘I-I— _— -.._v: . H‘DQer‘l‘ ‘ ' n 5.a&&yq‘~.~ biphasic Growth Medium..................... Studies on the Possible Possession of Arginase Activity by Coagulase...................... Possession of Phosphatase Activity=by Coagulase. The pH-dependence of the Coagulase and Phosphat- ase Reactions.............................. Thermal Inactivation Studies.................... Anion Exchange Chromatography................... Electrophoretic Studies......................... Studies on the Effects of Various Inhibitors on the Coagulase and PhOSphatase Activities... Saturation of Phosphatase Activity with Excess Substrate........................... Increases in Specific Activity of Coagulase and Phosphatase during Purification............ Studies on the Possible Possession of Lecithinase ACtiV‘lty by coagulasecoeoocoocccccccocoo... DISCUSSIONOOOOOOOOOOOIOOOOOOOOOOOOOOOOOOCOOOOOOOOOOO SWYOOOOOOOOOOOOCOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO BIBLIOGRAPHIC.OOOOOOOOOOOOOOOOOOOOOOOOOOOOCOOOOOOOOO iv 32 33 37 38 #1 43 46 49 53 57 57 61 66 68 .223 1. Effec 0; ceagc. 38- the 1; xi. A It - c. .32.: :1: coag 1a: LIST OF TABLES TABLE PAGE 1. Effect of various inhibitors upon the coagulase and phosphatase activities of the purified preparationOOOOOOOO0.000‘OOOOO... 52 2. Comparison of specific activities of coagulase and phosphatase during purification. 58 1.Conparis is. 7 selecte“ coccus ; neiia . . ‘ P :hcspna w- I l at. #1130»; e a. 3 5.1nsepa. phosph chrome and 0. 6.1flSEp: phCSp‘ tits: and C DKCSj elec bus: 8, 1286 Dh01 Ele buf \O p- r+ .4- ’c1 {3‘ n 0’“) ’4’ FIGURE 1. 3. 4. 5. 7. 9. 105 11 LIST OF FIGURES Comparison of growth of selected strains (3B, 7, 70) of Sta h lococcus aureus in biphasic and nonSIpEasIc media................ Comparison of see less production by selected strains BB, 7, 70) of Sta h lo- coccus aureus in biphasic and nonBIpEasIc me 3.0...OOOOOOOOOOOOOOOOOOOOOOOOOOOOCOOOOOOO The effect of pH upon coagulase and phosphatase 80t1V1ty...................o...... Comparison of the degree of thermal inactivation of coagulase and phOSphatase at 37 c and 56 o.O...O...OOOOOOOOOIOOOOOOOOOOO Inseparability of the coagulase and phosphatase activities by anion exchange chromatography employing DEAE-cellulose and 0.01 M tris buffer at pH 7.5.............. Inseparability of the coagulase and phOSphatase activities by anion exchange chromatography employing DEAE-cellulose and 0.01 M tris buffer at pH 8.600000900000000 Inseparability of the coagulase and phOSphatase activities by starch block electrophoresis employing 0.01 M tris buffer at pH 8.6.0O...OOOOOOOOOOOOOOOOOOOOOOOO Inseparability of the coagulase and phosphatase activities by starch block electrophoresis employing 0.01 M tris buffer at pH 7050000000000000050009.0000ococoo Attempt at separation of the coagulase and phosphatase activities by starch block electrophoresis employing a discontinuous buffer system; strip buffer composed of 0.1 M tris at pH 8.0 and.well buffer, 0.05 M barbital at pH 6.8..................... The velocities of the phosphatase reaction at various concentrations of substrate (p-nitrophenylphosphate) at 37 C........a..... Lineweaver-Burk plot of-phosphatase (p-nitro- phenylphosphate substrate) for the purpose of calculating the Michaelis constant......... v1 PAGE 34 35 42 45 47 48 50 54 56 ' r research to he stiles have ‘ relatively ur.‘ argriszs bec. between certs. being COEE‘AIEI sch sabstam erg-.nase hav ECCEpted a °f Pctent: Whit)“. Sta determine S‘llStancE Wild 01 INTRODUCTION The continuing emergence of complex therapeutic problems caused by Staphylococcus EEEEEE has stimulated a large amount of both clinical and physiological research to be conducted on this bacterium. Various studies have indicated that the staphylococci occupy a relatively unique position among pathogenic micro- organisms because of the supposed correlation existing between certain cellular substances, the most prominent being coagulase, and virulence. In addition to coagulase, such substances as phoSphatase, hyaluronidase, and arginase have been associated with the pathogenic capacity of staphylococcal strains. However, the exact role which these activities play in the establishment of a staphylococcal infection has yet to be elucidated. Coagulase has been assumed to be largely involved in the virulence of staphylococci and is generally accepted as the best single criterion for determination of potential pathogenicity. However, the mechanism by ‘which staphylocoagulase functions is not known. The determination of the biochemical activities of this Substance as well as its physical properties potentially could clarify its mode of action and even, perhaps, its :involvement in staphylococcal disease. An additional aspect would be to establish whether the correlations, Previously indicated to exist between coagulase and Other activities, were of a functional nature. To mm." AI .2! d h V .‘c e at“" ""‘-. o C "I'l:“f 0‘" "I 2 accomplish this end, the present investigation was undertaken. A . \Oai! ; qu-u ‘2: '.< ‘HP: lev V 2.... gib- . t" or! REVIEW OF LITERATURE Staphylococci and Coagulase One of the earliest descriptions of the ability of Staphylococcus §u£32§_to produce coagulase was reported by Loeb (1903) who demonstrated the in‘zitgg clotting of goose plasma. 'Using citrated human plasma Much (1908) found that pathogenic staphylococci exhibited the coagulation phenomenon. Conzenbach and Uemura (1916) observed the coagulation reaction in oxalated rabbit, goat and sheep plasma, as well as human plasma, with the rabbit plasma being the most coagulable. Later in 1926, DarEnyi, observing staphylococci from human and animal sources, recognized a correlation between the pathogen- icity of these microorganisms and their ability to clot plasma. He advocated that the clotting of citrated rabbit plasma was the best criterion in the separation of pathogenic from nonpathogenic staphylococci. Also finding that citrated plasma was the most suitable for determination of coagulase activity, Gross (1931) simil- arly concluded that coagulase production was indeed characteristic of pathogenic staphylococci. He also found that the potency of culture filtrates of virulent staphylococci paralleled the activity of the unfiltered cultures. Later authors such as Walston (1935). Fisher (1936) and Cruikshank (1937) confirmed the coagulase activity of culture filtrates. The production of coagulase and hemolysin by §. 3 4 agggug as criteria for virulence was examined by Chapman and his coworkers (1934). They found that out of 690 strains, 88% of these strains coagulated oxalated plasma whereas only 51.7% were hemolytic. They also showed that only 11.9% of 1,852 strains of Staphylococcus 31232 were coagulase-positive. The relatively lesser power of the‘§.‘glgg§'group to coagulate plasma as compared tol§. 235233 was also reported by other workers (Burnet, 1930; Fisher, 1936). Finding that nonhemolytic pathogenic strains of‘§. gagggg coagulated plasma and that, regard- less of pigment formation, strains which possessed coagulase were usually pathogenic, caused Chapman.g_,gl. (1934) to conclude that coagulase positivity is a valuable asset in conjunction with hemolysin production in the determination of a staphylococcal pathogen. Fisher (1936) also showed that coagulase-positive strains were nearly always hemolytic whereas coagulase- negative strains were most often nonhemolytic. Stephan (1935) reported that out of 45 staphylo- coccal isolates from pus, 37 (82%) were coagulase positive, whereas this was true only in 46% of the 45 isolates from blood, urine and sputum.. 0n the basis of his investigations and on the work of other authors, Cruikshank (1937) suggested that the coagulating ability of'§.'gg;gu§ represented the most convenient, reliable igggitgg method for the determination of the pathogenicity of a given.staphylococca1 strain whether it be of hits. such as Che} Javenish and Gristle, ii: toxin proiu agreed more iii coegilz Howe Sial‘fv'loco accepted 1 35 Blair reactions 5 it be of human or animal origin. Numerous authors such as Chapman 331 31. (1938), Gillespie _e_t 5;. (1939), Devenish and Miles (1939). Blair (1939). Christie and Keogh (1940), Fairbrother (1940) and Smith gt 5;. (1947) have confirmed and supported this criterion. However, there have been a few dissenting reports. Christie, North, and Perkin (1946) suggested that alpha toxin production by pathogenic strains of S. w agreed more closely with mouse pathogenicity tests than did coagulase production. However, at the present time, the ability of staphylococci to produce coagulase is generally accepted for the recognition of pathogenic strains and as Blair (1939) stated even though various in 3232.9. reactions such as hemolysis and pigment formation sometimes parallel the' coagulase reaction, the results are sufficiently varied to make them inadequate. Consequently Blair (1939. 1958) completely endorsed the use of the coagulase test as a sufficient single in 33:33.9. index of the pathogenicity of staphylococci. Various methods for the detection of coagulase- positive staphylococci have been preposed. The tube method approach, introduced by Loeb (1903). is fundamentally the inoculation of susceptible human or animal plasma with staphylococcal cells or their Supernatant fluids followed by incubation at 37 C. pylene glycol, sodium azide, and streptomycin F”? .A.1I~ 4 i'rbr. 15 inhibited partially purified staphylocoagulase whereas no inhibition occurred with penicillin, Zephiran, Tyrothricin, Bacitracin, Gramicidin, Tyrocidin, sulfathiazole, sulfadiazine and hydrazine sulfate. However, Miale (1949) found penicillin to be quite inhibitory towards coagulase. This discrepancy, of course, may be due to variations in the media employed and the method and degree of purification. Tager (1948) found that sodium thioglycollate (0.05% in saline) at pH 6.8 exerted no effect on coagulase while, on the other hand, cysteine retarded the deterioration of coagulase in saline. Ascorbic acid exhibited a strong inhibitory effect as did the oxidizing agent, superoxol. Cysteine was found to block the deleterious effect of the former compound. Crystal 'violet in a concentration of 1:100,000 and high concentrations of phosphate and borate ions have been reported to be inhibitory (Miale, 1949). Crude PreparatiOns of trypsin and pepsin (Walker _e_t 11., 1947) as well as crystalline preparations of the same PrOteolytic enzymes (Tager, 1948b) have been found to ina ctivate coagulase . . Relationship of Coagulase to other Biological Activities of Coagulase-positive Staphylococci Various biological properties of staphylococci have been investigated over the years in an attempt to differentiate the pathogenic organisms from the nonpatho- 16 genic ones. Since coagulase was early established as the index of pathogenicity, then the finding of another reliable criterion of pathogenicity might shed light on the mechanism by which coagulase functioned. The differentiation of pathogenic and nonpathogenic staphylococci by mannitol fermentation has been attempted by numerous workers. Hine (1922) found, as did Jullianelle (1937). that the fermentation of mannitol with acid production was indicative of pathogenic staphylococci. Hallman (1937) confirmed this observation in 90.9% of 487 strains. He also found agreement between the crystal-violet reaction of Chapman and Berens (1935) and the coagulase test in 88% of the strains tested. Similarly, Flaum (1938) reported a correlation between coagulation of rabbit plasma and mannitol fermentation, and.to a lesser degree, pigment production. Chapman 23 9;. (1938) attempted to determine the significance of 01‘ various biological tests for pathogenicity and found that the coagulase test was the most reliable single index whereas tests with crystal-violet agar, brom—thymol- blaze agar, mannitol fermentation and pigment production were useful only as supplementary tests. Differentiation of pathogenic and nonpathogenic 1"Yipes by different reactions in litmus milk was °r1ginally attempted by Dudgeon (1908) who observed no Significant correlation between the two types. Confirmation of this lack of correlation was made by 17 other workers such as Winslow _e_t _a_l.. (1920), Minett (1936) and Schlam and Woods (1953). In 1951 Barber 33 3.1. (1951) tested 160 coagulase- positive and 75 coagulase-negative strains of g. aureus for their ability to release phenolphthalein sulfate and phenolphthalein glucuronide. They reported that all 160 coagulase-positive strains liberated phenolphthalein from phenolphthalein phosphate whereas only 1 coagulase-- negative organism did so.) Neither type of staphylococci exhibited any significant activity on the other two substrates. Barber and Kuper (1951) also found that when 100 clinical staphylococcal isolates were incubated overnight on nutrient agar plus 0.01% phenolphthalein phosphate, after exposure to ammonia, 42 exhibited phosphatase-positivity while 58 did not. Colonies from the 58 negative plates proved to be coagulase-negative). When 160 representative colonies were picked from the 42 positive plates, all but one of these were coagulase- POSitive. Chiarolanza (1955) reported a strict relation- Ship between coagulating power and phosphatase activity. Lovell (1958) confirmed this finding and also devised a 80lid medium employing disodium p-nitrophenylphosphate as the substrate since he had experimentally showed its BuDeriority over other phosphatase substrates. 0n the other hand, Gupta and Chakravati (1954) round a doubtful relationship between coagulase and Phosphatase-positivity as did Mali]: and Singh (1960). 18 The latter authors reported that in addition to strains of §. aureus, some strains of1§. 31233 and g. citreus also exhibited phosphatase activity. The possibility exists that this discrepancy in results may revolve around the fact that the latter two articles were concerned primarily with staphylococci of animal origin. A relationship has also been suggested to exist between staphylocoagulase and arginase or an arginase-like enzyme. Fusillo and Jaffurs (1955) found that 23 of 36 organisms grown on arginine-yeast medium produced plasma clotting. They considered that arginine and arginase play an important role in the metabolic processes which form substances that activate the formation of fibrin. Another possible relationship to staphylococcal pathogenicity was reported by Hills (1940) who postulated the possible function of an arginine dihydrolase, which is distinguished from arginase by the fact that it catalyzes the formation of ammonia, carbon dioxide and glutamine from arginine, rather than ornithine and urea as does arginase. By following the growth of‘§.‘aureus on a special arginine medium by chromatography, Lominski 23‘21. (1952) reported that during incubation a citrulline spot occurred which increased in intensity with a corresponding decrease in the amount of arginine present. The presence of an active arginine, ornithine,. citrulline cycle was thus indicated. Therefore, as Fusillo and Jaffurs (1955) stated, these studies 19 suggested the possibility that coagulase might be related to an arginase or a similar type enzyme. Recently, Drummond and Tager (1959a, 1959b) have reported that purified preparations of staphylocoagulase possessed esterase activity upon tributyrin in addition to the normal coagulating activity. However,they (Drummond and Tager, 1959b) later achieved the physical separation of the coagulase and tributyrinase activities. Isolation, Concentration and Purification of Coagulase Early realization that an understanding of the mechanism by which staphylocoagulase functions in the plasma clotting mechanism could explain the relationship between coagulase and pathogenicity stimulated attempts on the concentration and purification of staphylo- . coagulase itself. One of these first attempts was carried out by Halston (1935). He precipitated coagulase from the filtrate of a broth culture of a coagulase- positive strain with 10 volumes of 96% alcohol. He also attempted precipitation with acetic acid and half- saturation with ammonium sulfate but found that alcoholic precipitation yielded a much more highly active coagulase. Also, the precipitate obtained by half-saturation with ammonium sulfate could not be completely dissolved in 'water. Fisher (1936) showed that the active principle could be isolated from cell-free filtrates by precipitation with 3 or 3% volumes of 95% alcohol. 20 According to Smith and Hale (1944) the thermostability of coagulase-containing filtrates allowed them to concentrate the filtrates by distillation at 60 to 70 C under reduced pressure. By this method they obtained a lO-fold concentratiOn together with some purification. They also suggested that partial purification could be achieved by alcoholic precipitation and dialysis. Walker £3 51. (1948) obtained his coagulase l. ~_J preparations by repeated precipitation with acid at pH 4. He initially autoclaved the coagulase-positive cultures for 20 minutes to remove interfering proteins, and after centrifugation, the supernatant fluid was adjusted to pH 4 with H01. The resulting precipitate was centrifuged in the cold. After washing with buffers of the same pH value but of progressively decreasing ionic strengths, the acid precipitation was repeated. The products obtained varied between 25 and 60 mg of coagulase containing from 4 to 10 mg of nitrogen. Tager (1948b) reported a procedure by which he Obtained highly purified coagulase preparations. Fifteen to 20-liter lots of crude coagulase in brain heart infusion broth were cleared of cells by a Sharples cBentr‘ifuge. The supernatant fluid was slowly acidified 1=0 pH 3.8 to 4.0 with 4 n HCl. After storage in the cold, the acid precipitate was collected by centrifugation, washed, redissolved in buffer, and then lplflecipitated with three volumes of 95% alcohol. This 21 alcohol treatment was repeated with intermittent precipitation of impurities with low concentrations of ammonium sulfate. The purified preparation was lyophilized and stored in a desiccator. It possessed high biological activity, clotting plasma in 3 or 4 seconds, and was found to be protein in nature. Purification of free coagulase from staphylococcal cultures in digest broth was described by Duthie and Lorenz (1952). They demonstrated that coagulase was readily precipitated by cadmium salts which could be removed by dialysis at a low pH. This procedure yielded a coagulase which was stable in solution at pH 2. Jacherts (1956) subjected this type of preparation to further purification by column chromatography, employing aluminum hydroxide, and paper electrophoresis. Duthie. and Haughton (1958) improved their purification method by obtaining a starting material which possessed a greater degree of purity than that usually obtained in culture ' supernatant fluids. They took advantage of the fact first demonstrated by Duthie (1954b) that if one volume of a mature shaken culture of‘§. aureus in digest broth 1 is inoculated into 9 volumes of temperature-equilibrated broth and is then shaken at 37 0, free coagulase is :maximally released within 120 minutes. In the present 'work, Duthie and Haughton (1958) employed a casein- hydrolysate medium and found a maximal ratio of coagulase to extracellular protein within 80 minutes. livtlllll‘ 1;l.1.t..l.l. 5 N o. .. .IIllii ..‘J o . .1— 22 Murray and Gohdes (1959b) purified coagulase principally by precipitation with.ammonium sulfate (33% weight/volume) and cadmium sulfate, followed by chromatography on a calcium phosphate column using stepwise elution with phosphate buffer. Recently, Blobel 25.31. (1960) reported successful purification based on acid precipitation of the original culture supernatant fluid followed by ethanol fractionation and electrOphoresis. MATERIALS AND METHODS Organisms and Culture Media In this study, 3 phage propagating strains of Staphylococcus aureus of the International-Blair series (Blair and Carr, 1953) were employed, namely 33, 7, and 70. These strains were experimentally determined to be representatives of staphylococci which produce a V relative low, medium and high amount of coagulase respectively. Strain 70 was the one which was purified and used for the subsequent studies in which purified preparations were employed. Stock cultures were maintained on brain heart infusion agar1 slants and stored at 4 C. ' In all experiments in which growth of g. aureus was required, an incubation temperature of 37 C was employed. Similarly brain heart infusion broth2 was constantly used as the liquid growth medium. Assay Methods Coagulase Coagulase activity was determined by a slight modification of the method of Tager and Hales (1947). In most cases, 1 ml of coagulase-containing solution was added to 1 m1 of a 2% peptone-saline solution (2 gm of peptone in 100 ml of 0.85% saline) containing 1’2Difco Laboratories. 139-: Detroit, Michigan 23 . (‘4'. 24 merthiolate at a concentration of 1:5000. After suitable dilutions were made, 1 m1 of a 1:5 dilution of standard- ized human plasma in saline was added to each tube,- thoroughly mixed, and incubated at 37 C. The final tube in which a clot was visible after 24 hr was taken as the endpoint. ‘ Phosphatase Procedure I. Phosphatase activity was determined by a modification of the method of Barnes and Morris (1957). The stock substrate solution employed was p-nitrophenylphosphoric acid, disodium salt‘ at a concentration of 0.005 gm/ml and was stored at 4 C until needed. Usually a l-ml sample was placed in a standardized cuvette (12 mm by 100 mm) containing 1.4 m1 of tris buffer, pH 7.2. To this was added 0.6 m1 of stock substrate solution. After incubation in a water bath at 37 o for 30 min, 3 ml of 0.01 N NaOH (pH 12) was added to stOp the reaction and to completely develop the color of the liberated p-nitrophenol. Optical density of the alkaline solution was determined using a Bausch and Lomb Spectronic 2O Spectrophotometer at a wavelength of 400 mm. A control tube consisting of buffer and substrate was treated similarly. By comparison of the intensity of the color produced to that of a standard curve of p-nitrophenol, the activity 1Aldrich chemical 00., Milwaukee, Wisconsin 25 of the enzyme in terms of pH p-nitrophenol liberated was obtained. Procedureigg. For the measurement of‘the amount of inorganic phosphorous released, a modification of Allen's method (1940) was employed. To a 2-ml sample was added 8 ml of 10% trichloroacetic acid and the precipitate thus formed was millipore filtered. To the protein-free filtrate was added 2 ml of 10 N H2804, 2 ml of monomethyl-parasaminophenol sulfate (Piotol), 1 m1 of ammonium molybdate, and 10 ml of distilled water to bring the total volume to 25 ml. Incubation at room temperature for 15 min was followed by spectrOphotometric measurement at 625 mp. eThe amount of phosphorous released was then determined via a previously devised standard curve . Protein Protein was determined by the Folin-Ciocalteu reagent (Lawry'gt'gl., 1951). One ml of l N NaOH was added to a l-ml sample, mixed well and incubated at room temperature for 10 min. To this was added 1.5 ml distilled water and 5 ml of Reagent A (1 ml of 2.7% sodium potassium tartrate .4 H20 and 1 ml of 1% copper sulphate .5 H20 added to 100 m1 of 2% sodium bicarbonate). After standing at room.temperature for 15 min, 0.5 m1 of Reagent B (commercial Folin-Ciocalteu reagent) was added, mixed immediately, and stored at room temperature for 25 min. Optical density readings were made at 660 mu 26 and protein content determined from a standard curve for bovine albumin, fraction V.) Isolation and Purification of Coagulase Six tubes, each containing 10 m1 of broth, were inoculated from a brain heart infusion agar slant culture. After incubation for 18 hr, 5 ml of each of the above tubes were correspondingly inoculated into six SOO-ml Erlenmeyer flasks, each containing 100 ml of broth, and incubated for 7 hr. This logarithmic-phase growth was in turn used to inoculate a total of 6.6 liters of broth medium using 6-liter Florence flasks each containing 3.3 liters. The organisms were incubated for 12 hr on a rotary shaker (150 cycle/min). The organisms were removed using a Servall continuous flow, superspeed centrifuge2 at a flow rate of 100 ml/min. The coagulase in the supernatant fluid was purified principally by the methods developed by Tager (1948b) and more recently modified by Blobel 23 31. (1960). Since some procedures and reagents were further changed or modified, the actual process employed will be briefly outlined. The supernatant fluid was slowly adjusted to pH 3.8 by 4 H H01 producing a precipitate of coagulase. This precipitation process was continued at 4 C for 20 hr. The acid precipitate was then collected by 1Nutritional Biochemicals Corporation, Cleveland, Ohio 2Ivan Sorvall, Inc., Norwalk, Connecticut ‘F._‘?.—‘ f'fTTfiT‘Tm—u' a an.” 27 continuous flow superSpeed centrifugation, dissolved in 50 m1 of distilled water and adjusted to pH 7.2 with 0.066 M disodium phosphate buffer. After thorough mixing in the cold, the insoluble residues were removed by a centrifugal force of 12,100 x G for 20 minutes. The cooled supernatant fluid was then precipitated by adding 95% ethanol kept at -20 C to a final ethanol concentration of 70% (v/v). The recommendations by Cohn and his coworkers (1946) for alcoholic precipitations were observed by maintaining the environment at a temperature not higher than -5 0. Also, the precooled ethanol was added very slowly through a syringe needle with constant stirring. This precipitation was allowed to continue for 20 hr after which time the resulting precipitate was collected by centrifugation at 1,935 x G at -20 C. The alcoholic supernatant fluid was discarded, and the precipitate was resuspended in 50 ml of distilled water and adjusted to pH 7.2 with 0.066 M potassium dihydrogen phosphate buffer. After thorough mixing, the insoluble :residues were again removed by centrifugation and the supernatant fluid subjected to another complete cycle of ethanol precipitation. This final clear solution was concentrated about 4-fold by dialysis against polyvinyl- pyrrolidine1 for 24 hr at 4 C. The resulting coagulase preparation was then 1y0philized and stored at -20 0. 1Oxford Laboratories, Redwood City, California 28 Anion Exchange Chromatography N,N-diethylaminoethylcellulose (DEAE-cellulose) was thoroughly washed with 0.01 M 2-amino-2-hydroxymethyl-1, 3-pr0panediol (tris) buffer at pH 7.5 for 16 hr. This slurry was poured into a glass column (30 cm in length and 1 cm in diameter), and allowed to settle, maintaining a head of buffer to prevent drying out of the adsorbent. The adsorption column was then packed to a height of 18 cm by air pressure, washed well with buffer at 4 C to obtain pH and temperature equilibrium, and set up over a fraction collector). Thirty mg of lyophilized purified coagulase, which had been dissolved in 1 m1 of 0.01 M tris buffer pH 7.5 and dialyzed against the same buffer for 24 hr at 4 C, was introduced into the column. The coagulase was then subjected to gradient elution with the reservoir containing 1.25 M Na01. The flow rate was such that 5 ml fractions were collected every 10 minutes. Elution was continued at 4 C for approximately 15 hr and a.total of 76 fractions was obtained. Each fraction was assayed for coagulase and phosphatase activity as well as protein. The same general procedure was followed when buffers of other pH values were used. Electrophoresis Starch block electrophoresis The following general procedure was followed for 1Rinco Instrument 00., Greenville, Illinois 29 the complete series of starch block electrophoretic experiments. In certain individual experiments various buffers of different pH values were employed, which will be indicated in the appropriate sections. In order to remove any protein impurities, insoluble potato starch was washed three times with SOO-ml amounts of 0.02 N KOH. This treatment was followed by similar washings with three 500-ml amounts of distilled water and finally with the appropriate buffer. The starch was then molded into a block, 24 cm by 2 cm, by means of‘a plastic template. By careful blotting with highly absorbent paper, excess buffer was removed without disturbing the surface of the settled starch granules. Twelve mg of the lyophilized coagulase preparation, which had been dissolved in 0.3 m1 of apprOpriate buffer and dialyzed for 24 hr at 4 0 against this same buffer, were applied to the block by excising a 2 mm segment of starch and replacing it with a slurry of coagulase and starch granules. The starch block was placed in a leveled horizontal position between two plastic electrolytic containers enclosed within a plastic box prior to equilibration to 4 0. Heavy filter paper strips were used as the electrolytic contacts. The material was then subjected to 200 v for 16 hr with an average current of 2 ma. At the end of the electrOphoretic run the starch block was carefully segmented at 1-cm intervals, placed in separate tubes and resuspended. Upon settling of the starch granules 30 the contents of each tube were assayed for coagulase and phosphatase activity. Protein content was also determined in certain instances. Starch gel electrophoresis: The starch gel used for this type of electrophoresis was prepared according to the procedure of Smithies (1955). Hydrolyzed starch1 (6.24 gm) was dissolved in 50 m1 of 0.01 M tris buffer at the desired pH values. The gel was heated through its semi-solid state to a viscous liquid, quickly degassed by negative pressure, and poured onto a plastic template, 24 cm in length by 2 cm in width. After setting of the gel, a 2-mm segment was removed and replaced by a mixture of coagulase (l2 mg/0.3 ml buffer) and starch granules. White petrolatum was melted, cooled, and layered over the surface of the gel to prevent evaporation. Strips of filter paper served as contacts between the starch gel block and the buffer in the plastic electrolytic vessels. The coagulase preparation was subjected to 200 v resulting in a current of 2 ma. One-om segments were removed, placed in 5 m1 of distilled water, and homogenized with a glass pestle. After settling of the homogenized gel, sometimes aided by low speed centrifugation, each of the supernatant fluids were assayed for coagulase and phosphatase activity. 1Connaught Research Laboratories, Toronto, Ontario 31 Paper electrophoresis The horizontal open strip method of paper electrophoresis was conducted at 4 C. Whatman 3 MM paper strips, 47 mm wide and 32 cm in length were employed. The paper strip was dampened with 0.01 M tris buffer at a given pH, stretched taut between the electrolytic vessels and equilibrated to 4 C. By means of a micro- pipette, 25 x 10"4 ml of coagulase (l2 mg/O.3 ml) in buffer was applied in a straight line across one end of the paper strip. After subjection to 200 v and a current of 1 ma for 16 hr, the strip was cut into 2 equal portions lengthwise and at l-cm intervals crossways. Consequently, 2 equal portions of the paper strip for each centimeter distance in the migration path were obtained. One of these was eluted with 1 ml of distilled water and assayed for coagulase while the other corresponding portion was similarly eluted but tested for its phosphatase activity. RESULTS Coagulase Production in a Biphasic and Nonbiphasic Growth Medium We have previously found in our laboratory that brain heart infusion broth is a suitable medium to yield a maximum amount of staphylococcal growth. In order to obtain initially as large an amount of coagulase as possible, the value of a biphasic growth system over that of a nonbiphasic system was investigated. Tyrrell gtfigl. (1957) reported that yields up to 6x1011viable organisms per ml were obtained with a biphasic system consisting of a layer of solid nutrient medium overlaid with nutrient broth. With this system an increase in the concentration of cells of 2- to 30-fold was obtained without sacrificing total yield. In our studies, three strains of g. aureus (3B, 7, 70) were used. Logarithmic phase growth from each of these three strains was spectrophotometrically standardized to 0.05 OD and used as inoculum. The biphasic system consisted of 100 ml of broth over a layer of 50 ml of solidified brain heart infusion agar in a BOO-ml flask. The nonbiphasic system consisted of 50 m1 of broth alone. After inoculation the flasks were incubated on a rotary shaker (150 cycles/ min). At various intervals of time, samples were removed and growth determined by means of optical density at 650 my. The coagulase activity of the supernatant fluid of the same growth sample was also determined. The 32 33 biphasic system began to show superior growth over the nonbiphasic system within 8 hours and after 24 hours the difference in growth was quite pronounced (Fig. 1). For example the increase in growth of strain 70 grown biphasically compared to strain 70 grown nonbiphasically 'was 4.5-fold as measured by viable cell counts. These results agree with the fundamental hypothesis that the biphasic system by continued elution and adsorption of growth-enhancing substances and inhibitory cell products will result in the production of a greater concentration of cells. However, the amount of coagulase produced by each strain grown in either type of growth system was quite similar (Fig. 2). It was also found that the maximum production of coagulase was followed by a decrease in coagulase activity. In the case of strain 3B, it was more pronounced than with strain 70. It was also observed in both growth systems that maximum coagulase production was obtained prior to maximum growth of cells. A similar observation was indicated by Duthie and Haughton (1958) for their growth system. It was, therefore, concluded that brain heart infusion broth supplies a satisfactory medium for the production and isolation of a maximum amount of coagulase. Studies on the Possible Possession of Arginase Activity by Coagulase It has previously been suggested that a correlation exists between the coagulase and arginase Optical Density at 650 mp 34 I2 n.70 _ 417 AH3B l0" - A—A Biphasic Medium ‘ 8 — 0—0 Non- Biphasic Medium 7 O \' /1 1 1 i i 1 1 1 1 6 l2 I8 24 Hours at 370 Fig. 1. Comparison of growth of selected strains (3B, 7, 70) of Sta h lococcus aureus in biphasic and nonbiphasic media. 35 524,288 , A—A Biphasic Mednum c—c Non- Biphasic Medium 65,536 __ /\x 70 ‘3 :2 8H92 '— I"? e i i7 .9- 8 U024 (I o: a» o ‘5 8’ l28 A. :::::: o 0 A 16 . 38 O 1 1 1 1 J, 1 1 1 6 |2 18 24 Hours at 37 C Fig. 2. Comparison of coagulase production by selected strains (3B, 7, 70) of Sta h lococcus aureus in biphasic and nonbiphasic meEi . 36 activities of _s. M (Fusillo and Jaffurs, 1955). If a correlation did exist, it might be due to (1) a chance relationship between two biochemically unrelated phenomena, or (2) a biochemical relationship function- wise. To resolve the basis of this correlation, standard manometric procedure (Umbreit 23.51., 1957) was used. The main compartments of one set of exogenous Warburg flasks each contained 1 ml of 0.05 M phosphate buffer (pH 4.8) and 1 ml of purified coagulase solution in the same buffer (0.5 mg/ml). The side-arms each contained 1 m1 of arginine (0.005 M). Another set of exogenous flasks contained, in addition to the above, 0.2 m1 of 20% KOH in the center well to absorb any 002 present. After equilibration for 15 min at 37 c, the stopcocks were closed and measurements begun. No significant 02 uptake or evolution of gas occurred. Since most arginases are activated by Mn++ and generally have an optimum pH of 9-10, another series of manometric experiments were conducted employing 0.05 M phosphate buffer of pH 9 and 0.001 M Mn*+, as well as enzyme preparation and substrate. Similar studies were also carried out in an atmosPhere of 5% 002 and 95% N2. Where necessary, double side-armed flasks were employed with one of the side-arms containing 1 ml of 0.1 N H01 which was tipped into the main compartment at the end of the manometric run in order to release any 002 adsorbed. However, no significant 02 uptake or gas 37 evolution was observed in any of the reaction vessels. Employing Nessler's reagent, the reaction mixtures from these manometric studies were found to possess no NH}. They were also found to contain no urea as determined spectrophotometrically at 475 my using Hycel 2, 3- butanedione reagent). The contents of representative exogenous flasks were treated by paper chromatography. Samples of 0.05 ml were spotted on Whatman #1 paper and water-saturated phenol was used as solvent. After 12 hr, the chromatogram was developed by Spraying with a 0.1% alcoholic solution of ninhydrin followed by heating at approximately 80 0. Only one spot was observed which, by comparison to a control, was identified as arginine, the original substrate. Consequently, even though the coagulase preparation was provided with a specific cofactor and Optimum pH for arginase enzyme, no indication either manometrically or chromatographically was found to suggest that purified staphylocoagulase possessed an arginase or an arginase- like activity. Possession of PhOSphatase Activity by Coagulase The investigations of various workers have indicated that a correlation exists between the coagulase and 1Scientific Products, Evanston, Illinois 38 phOSphatase activity of staphylococci. Preliminary studies showed that the purified coagulase preparation from §, aureus phage prOpagating strain 70, possessed a concomitant phosphatase activity as measured Spectro- photometrically at 400 mp using p-nitrophenylphosphate as the substrate. Since the actual mechanism by which staphylocoagulase clots citrated plasma is poorly understood, the finding of a significant phOSphatase activity in a purified preparation of coagulase was of considerable interest. In order to clarify this dual phenomenon a series of investigations consisting of various approaches was conducted. These approaches were intended to determine (1) whether the same active group on a common protein accounted for both activities; (2) whether different active groups were associated with the same protein entity; or (3) whether two proteins, intimately associated, each possessed a different active group. The pH-Dependence of the Coagulase and Phosphatase Reactions The pH-dependence of enzymic reactions is usually a critical function. In these studies the staphylo- coagulase and staphylophosphatase reactions were performed at different pH values within the range of pH 5.1 to 8.8. Since no one buffer can be employed over such a pH range, two different buffer systems were used, namely, citrate and tris. It is well known that certain 39 ionic constituents of various buffers activate or inhibit enzymatic systems, sometimes skewing the pH- dependence curve. Consequently, the use of phOSphate buffer was avoided since phosphate ions have been reported to inhibit acid phOSphatase (Roche, 1950). Citrate buffer was used within the limits of pH 5.1 to 6.7, whereas tris buffer was used for the pH range of 7.2 to 8.8. The concentration of coagulase preparation in the reaction tubes was 0.5 mg per tube. As seen in Fig. 3, the coagulase and phosphatase reactions have the same optimum pH (pH 7.2). However, the coagulase was more sensitive to pH values on both the acid and alkaline side of pH 7(2. The fact that both reactions occurred optimally at the same pH might not be of possible significance. The basis for this conclusion was that the two reactions under study were involved with two different substrates whereas it is well known that even one enzyme might exhibit different pH optima on different substrates. When the effect of pH on the rate of an enzymatic reaction is studied, one of the major factors to consider is the stability of the enzyme. In these experiments, the influence of pH on the stability of the enzymes was not differentiated from the pH-dependence of the actual catalytic reactions. Therefore, the results presented in Fig. 3 represent the total effect of the various factors which contribute to pH-dependence curves such 4O ICC-— 0—0 Coagulase A—A Phosphatase A\\\ 80-— 2? A E 23 < '5 60)— .5 X g o “5 394iir' ‘ A O 20 *- ‘/‘ / ' A ’,/”////’ O 1 1 1 1 5 6 7 8 9 pH Fig. 3. The effect of pH upon coagulase and phosphatase activity. 41 as the degree of dissociation of enzyme protein and substrate, and enzyme stability. The elucidation of these pH optima as they existed under standard test conditions determined the pH at which future phosphatase and coagulase assays were conducted when maximum activity was desired. Thermal Inactivation Studies Thermal inactivation studies were carried out on the phosphatase and plasma clotting activities of purified coagulase in order to differentiate the two substances by their susceptibilities to heat. Samples of coagulase preparation (0.5 mg/ml) were placed in thermoregulated water baths at 37 0 and 56 0. After % hr, 2% hr, 5% hr, and 9 hr, samples were removed, immediately cooled and assayed for coagulase and phosphatase activity. The effects of these temperatures on the two activities were then compared (Fig. 4). It is apparent that the curves for the two activities appear quite similar with parallel decreases in both. After heating at 37 0 for 4 hr, the residual activity of the coagulase was 85% and that of the phOSphatase, 83,2%. Similarly, the preparation after heating at 56 0 for 5% hr retained coagulase and phosphatase activities of 12.5% and 7.4% respectively. This similarity in thermal inactivation suggested that the same protein entity was involved with both activities. ‘7. Residual Activity 42 IOOI‘ i\ \. ‘\\\-; 37c 80 t . c Coagulase A—A Phosohaiase 6O ' 4C)- 0 56C 20 " ‘\ \. A I 2 3 4 5 6 Time (hours) of Heating Fig. 4. Comparison of the degree of thermal inactivation of coagulase and phosphatase at 37 C and 56 0. 43 Anion Exchange Chromatography Anion exchange chromatography was employed in an attempt to determine whether or not the coagulase and phosphatase activities were physically separable. A DEAE-cellulose column was prepared using 0.01 M tris buffer. Lyophilized coagulase preparation (30 mg/ml) was applied to the column, gradiently eluted with sodium chloride and the effluent collected in fractions each of which was assayed for coagulase and phosphatase activity as well as protein. Fig. 5 indicates the results obtained at pH 7.5 with the eluate collected in S-ml fractions at the constant rate of 5 ml/lO min. A total of 76 fractions were collected. The two activities occurred maximally in the same fraction. A single protein peak emerged simultaneously with the two activities and the maximum peaks of activity for both the coagulase and phosphatase expressed in terms of specific activity (units of enzyme activity per pg of protein) also coincided. When chromatography was carried out in the same manner but at pH 8.6, similar results were obtained (Fig. 6). The two activities again showed similar maximum peaks of activity with remarkably sharp resolution. The obvious decrease in the activities of the fractions collected at pH 8.6 as compared to pH 7.5 'was due to the lesser stability of both substances at the higher pH and the one-third decrease in concentration 44 l l024 Coagulase Reciprocal Tilers 4.0 e [.0 ‘ 5|2 v \ ‘3 r ‘r' “.0 1 256 a) .. \ :1 3.0 ~ 1 I» 0 0 Coagulase 1 I28 8 l ‘ A A Phosphatase c 2 ' 4 64 3 I l ’2: 2.0 t- I W a 32 a 2 ‘ l6 1 1 ‘ ID i- n A \ q 8 ‘\ Au « 4 g A L‘ l ‘\ 1 1 L l IO 20 30 4O 50 60 70 Fraction Collected (tube number) Fig. 5. Inseparability of the coagulase and phOSphatase activities by anion exchange chromatography employing DEAR-cellulose and 0.01 M tris buffer at pH 7.5. 45 0.4 _. Coagulase Reciprocal Titers I — '0 _ -*256 .2 o 503% m -me .J '5 — ”- 0—0 Coagulase — 64 5 PM .5 <3” 02 _ I o A—A Phosphatase _ 32 32 c3. - l6 :2 1‘ -‘8 —‘4 1 l l 1 I I0 20 30 4O 50 60 7O Fraction Collected (tube number) Fig. 6. Inseparability of the coagulase and phOSphatase activities by anion exchange chromatography emplaging DEAE-cellulose and 0.01 M tris buffer at pH .6. 46 of coagulase preparation originally applied to the column. Electrophoretic Studies A complete series of electrophoretic studies was carried out on the purified coagulase preparation to determine if the coagulase and phosphatase were separable. If one faund that the two activities showed similar maximum peaks of activity after migration, then one could conclude that the two activities were electro- phoretically indistinguishable. The results of a typical experiment employing insoluble starch as the stabilizing medium.and 0.01 M tris buffer at pH 8.6 are shown in Fig. 7. At the end of the electrophoretic run the block 'was segmented, resuspended, and upon settling of the starch granules, coagulase and phosphatase determinations ‘were made. It is evident that both the coagulase and phosphatase possessed similar maximum peaks of activity after migration. The electrophoregram thus indicates V that the two activities are associated with the same protein under the given experimental conditions. Fig. 8 indicates that similar results were obtained when electrophoresis was carried out using the same buffer at pH 7.5. The preparation moved the same distance but in the opposite direction, showing that the isoeleetric point was between pH 8.6 and pH 7.5. Other electrophoretic studies involving different buffer systems, pH values, and stabilizing media were 47 F ‘ 5|2 - 0 0 Coagulase A Phosphatase 0.9” A. “'28. \ 0.6 - o o 32 1 pM p- Nitrophenol Liberated / Coagulase Recuprocal Titers l l l 8 IO l2++l L l 1 L 0-l4 2 0 2 ‘4 cm. Fig. 7. Inseparability of the coagulase and phOSphatase activities by starch block electrophoresis employing 0.01 M tris buffer at pH 8.6. 4.0 _ 0 ~ 5|2 0—0 Coagulase P B .—. Phosphatase A 2: an .e. 3 __ pH'lS . O 5 g 2.0 - A 32 2 E l. a / 2 LC - . l ~ 8 .1 A l L L I I’l‘ l l 1 HQ l0 8 6 4 2 O 2 4(+l cm. Fig. 8. Inseparability of the coagulase and phosphatase activities by starch block electrOphoresis employing 0.01 M tris buffer at pH 7.5. Coagulase Reciprocal Titers 49 conducted. Electrophoresis was carried out using barbital buffer as well as tris and at pH values from 4.2 to 10. The stabilizing media employed included paper’and starch gel. A total of 20 electrOphoretic determinations were made with the same results being obtained, thus suggesting that the same protein was involved with both activities. The coagulase preparation was also subjected to electrOphoresis using a discontinuous buffer system (Goldberg. 1959). The same procedure described for starch block electrophoresis was employed except that the starch block buffer was 0.1 M tris (pH 8.0) whereas the well buffer was 0.05 M barbital buffer (pH 6.8). Electrophoresis was carried out at 50 v for 20 hr with an average current of 4 ma. Under these experimental conditions a small degree of separation of the two activities seemed to occur (F18. 9). However, it was not possible to recover appreciable amounts of either activity in the complete absence of the other, because of the large amount of overlapping of the two activities. If a true separation of the two activities was obtained, then the fact that it was of such a small degree after treatment with a very critical electrophoretic system reemphasizes the probable similarity of the two activities. Studies on the Effects of Various Inhibitors on the Coagulase and Phosphatase Activities The effect of certain enzyme inhibitors upon the 0—0 Coagulase A—i Phosphatase 4r)- 0 “ -EN92 o/ \5 ° l .2 E :3 3-0 - . — 1024 L] ‘\\ '6 C d) o E E: 2()-- -l28 32: A . O Q 12 1 L0 b —l6 A 0—0 x 1 44:;r4N’A/1 1 1\\ 1 1 (-l l0 8 6 4 2 O 2 4 (H 50 cm. Fig. 9. Attempt at separation of the coagulase and phosphatase activities by starch block electrophoresis employing a discontinuous buffer system; strip buffer composed of 0.1 M tris at pH 8.0 and well buffer, 0.05 M barbital at pH 6.8. Coagulase Reciprocal Titers 51 two activities of the purified coagulase preparation was investigated. If coagulase and phosphatase were the same catalytic substance, then the degree of inhibition exerted by these compounds on each activity would be the same. On the other hand, if the preparation possessed two different catalytic groups, then different degrees of inhibition could occur. The inhibitors employed were ethylenediaminetetraacetate (EDTA), sodium fluoride, sodium azide, sodium.iodoacetate, and p-chloromercuriben- zoate (Table 1). A 2.5-m1 sample of coagulase solution (0.5 mg/ml) was incubated with an equal volume of each inhibitor. After incubation at 37 G for 120 min, samples were removed and coagulase and phosphatase determinations made. The controls consisted of coagulase at the appropriate concentrations in the absence of inhibitors. It was apparent that the degree of inhibition caused by each of the compounds was different, suggesting that the two activities were not caused by the same functional group. It also seemed that sulfhydryl groups were involved, particularly with respect to the phosphatase since it was inhibited 97% by the p-chloro- mercuribenzoate. The inhibition by iodoacetate further confirmed this interpretation. Sodium fluoride, a metal inactivator, and EDTA, a chelating agent, were especially inhibitory to the phosphatase. However, disregarding these other facts of interest, the main conclusion to be derived is that the two 52 TABLE 1. Effect of various inhibitors upon the coagulase and phosphatase activities of the purified preparation Final Per Cent Inhibitor Cone. Residual Activity ' (M) Coagulase Phosghatase EDTA* 2.5 x 10"2 50 2 Iodoacetate 2.5 x 10"2 50 76 Fluoride 5.0 x 104‘ 50 26 Azide 5.0 x 10"1 50 60 p-Chloromer- _2 curibenzoate 2.5 x lO 50 3 *Ethylenediaminetetraacetate ' 53 activities do not function by the same mechanism. Saturation of Phosphatase Activity with Excess Substrate The supposition that the coagulase and phosphatase activities are not caused by the same functional group was verified by the failure to inhibit coagulase activity when the phosphatase was saturated with excess substrate. This experiment was accomplished in the following manner: into 5 cuvettes there was placed p-nitophenylphosphate so that the cuvettes contained a final concentration of 1.92:3.0“2 M. 9.5x10'3 M, 4.75:10’3 M, 2.38::10"3 M, 1.19x10‘3 M respectively. To each of these cuvettes was added 2.4 ml of purified coagulase (0.5 mg/ml). Controls consisted of the appropriate concentration of substrate to which 2.4 ml of distilled water was added instead of enzyme. The reaction tubes and controls were incubated in a water bath at 37 C. At various periods of time, the phosphatase activity of the cuvettes was determined spectrophotometrically at 400 my, using the appr0priate controls as blanks. Consequently the velocities of the phosphatase reaction at different concentrations of substrate were obtained (Fig. 10). From these data the values for.§ (S=substrate concentration, v=velocity of reaction) at the various concentrations of p-nitrophenylphosphate substrate were calculated. As cited by Wilson (1950) the typical Michaelis-Menton equation of K3 = I¢§_ -S may be 54 L2— BDmM 95mM LO 4JSmM d. E O 0 QB d- ‘6 > E g 05 258mm 0 ‘6 2 ‘a 00.4 HSmM 02 l l I J l l L l 1 IO 20 30 4O 50 60 7O 80 Time (minutes) Fig. 10. The velocities of the ph03phatase reaction at various concentrations of substrate (p-nitrophenylphOSphate) at 37 C. 55 rearranged to the linear equation, _S_ = _1_ .S + 53 . A v V Lineweaver-Burk plot was made (Fig. 11); that is,'§ was v plotted as the ordinate and S as the abscissa. The slope of the resulting straight line is‘% and the ordinate intercept, gf. Thus the value for the Ks of the phosphatase reaction was calculated to be 1.599 x 10‘3 M. In order to saturate the phosphatase of the purified coagulase preparation, 2.4 m1 of the preparation (0.5 mg/ ml) was placed in a cuvette containing 100 times the K3 value of phosphatase substrate. A control consisting of the same concentration of substrate without enzyme was also prepared. The cuvettes were incubated at 37 C and the reaction allowed to proceed to completion as determined spectrophotometrically and by measurement of the release of inorganic phosphorous. The coagulase activity of the coagulase preparation was then determined and found to be completely intact. The fact that the phosphatase was completely satur- ated even in the presence of plasma substrate was verified by the complete lack'of liberated inorganic phosphorous during the coagulation of the plasma by the saturated preparation. Phosphorous was released from plasma, however, when the phosphatase was not saturated with p-nitr0phenylphosphate. It may be concluded therefore, that the two activities of the purified preparation do not represent the same functional groups. 56 (18 P 06 .0 .n C) n: Substrate/ Velocity 1 1 ' 1 1 1 4 8 l2 l6 20 Substrate Concentration (mM) Fig. 11. Lineweaver-Burk plot of phosphatase (p-nitrophenylphOSphate substrate) for the purpose of calculating the Michaelis constant. 57 Increases in Specific Activity of Coagulase and Phosphatase during Purification Another approach was carried out to determine if the two activities were associated with the same protein. If two different catalytic groups resided on the same protein entity, then.during the process of purification, one should find parallel increases in activity on the basis of protein. Consequently, coagulase was isolated from _§. aureus strain 70 and purified as previously described. After each stage of purification, the material was assayed for its coagulase and phosphatase activity and protein content. The results obtained during the progressive purification steps of the coagulase and phosphatase are seen in Table 2. Examination of the specific activity of both activities revealed a parallel increase after each purification step. The evidence again suggested that the coagulase and phosphatase were associated with the same protein entity. Studies on the Possible Possession of Lecithinase Activity by Coagulase Drummond and Tager (1959a) reported Tager's earlier observation that when incubated with egg yolk, purified staphylocoagulase produced a turbidity resembling that formed by the toxin (lecithinase) of Clostridium perfriggen . Chu (1947) (showed that Bacillus 223239 was capable of causing opacity in egg yolk medium due to its lecithinase. Staphylococci also produce this opacity (GilleSpie and Alder, 1952). Since 58 TABLE 2. Comparison of specific activities of coagulase and phosphatase during purification. _LA A Purification Specific Activity Fold Increases Step+ in Activity 1 E i Coagulasef Phosphatase** Coagulase Phosphatase L ' (x 10'3) i g A 0.70 0.85 B 3.23 3.38 -—X 4.6 4.0 0 3.00 3.18 f- 2.5 2.9 J D 7.87 9.80 2.7 3.1 E 8.09 10.0 .2 F 15.33 18.0 1.8 1.8 G 36.10 32.0 2.3 1.8 +A Initial broth supernatant fluid; B = Acid ppt, pH 3.8; C = Redissolved acid ppt from which insoluble residues removed by centrifugation; D = let ethanol ppt; E ist ethanol ppt, redissolved, and insoluble residues removed by centrifugation; F = 2nd ethanol ppt; G = Lyophilized, dialized residue. *Reciprocal titer/pg protein **pM p-nitrophenol liberated/pg protein 59 purified coagulase preparations were found, as reported in this thesis and elsewhere (Inniss and San Clemente, 1961), to possess a concomitant phoSphatase activity, it seemed reasonable that the action on egg yolk might be due to this phosphatase activity. Consequently, studies were conducted to determine the possible lecithinase activity of the coagulase preparation. The lecithinase of 2;. welchii andIB. cereus split lecithin into acid- soluble phosphorylcholine and the rate of increase of acid-soluble phosphorous served as a convenient measure of the enzyme activity. In the present investigation lecithinase was measured by determination of the increase of acid-soluble phosphorous. The method employed was essentially the same as that used by Macfarlane and Knight (1941) and Chu (1949). The reaction mixtures consisted of 1 m1 of purified coagulase (0.5 mg/ ml), 1.4 ml tris buffer pH 7.2 and 1.6 ml of a 3% lecithin emulsion. Controls consisted of coagulase without lecithin substrate and substrate alone. After incubation at 37 C for 30 min, the reaction was terminated by the addition of 20% trichloroacetic acid which also flocculated the unused lecithin. The contents of the tubes were then filtered through millipore filters and the total phosphorous in the filtrates was determined (Umbreitsgt‘gl., 1957). N0 increase in total phosphate was found over that of the controls. The coagulase-phosphatase preparation, therefore, possessed no lecithinase activity. 60 DISCUSSION One of the initial requirements for these invest- igative attempts at elucidation of the biochemical activities of staphylocoagulase was the production, isolation and purification of the clotting activity. The first step in the production of a coagulase preparation was the selection of a strain of §.‘§ggggg which possessed the ability to produce a large quantity of coagulase. A member of the International-Blair series of phage propagating strains (Blair and Carr, 1953) was considered desirable since considerable data had been previously obtained about these organisms in our laboratory and consequently the possibility was ever present that some fruitful relative information might develop. Preliminary investigations revealed that phage prOpagating strain 70 was the organism which best fulfilled this criterion. The optimal conditions for the production of coagulase in relatively large quantities were also investigated. A biphasic growth system (Tyrrell gtflgl., 1958) of brain heart infusion composition gave a greater yield of organisms than that obtained from growth in a :nambiphasic system of brain heart infusion broth. However, the latter system produced the same amount of coagulase and.since it possessed the added advantage of ease of preparation, it was employed. Shaken cultures were 61 62 employed to provide sufficient aeration and to facilitate release of the free coagulase into the growth medium. Under the cultural conditions used it was also observed that coagulase production was approximately proportional to growth up to 12 hours but after that time some loss of activity occurred even though growth continued. Consequently, the coagulase was harvested after 12 hours of growth. As previously indicated, the isolation and purifica- .tion procedures employed were essentially those developed by Tager (1948b) and more recently modified by Blobel gt 2;. (1960). However, the use of ammonium sulfate as suggested by the former author was avoided since it has been reported to be unsuitable for the removal of impurities from coagulase solutions (Blobel gt_§l.. 1960). The actual mechanism by which staphylocoagulase coagulates plasma is not known. Consequently, the finding of phosphatase activity in the purified preparations of coagulase suggested a new concept as to its biochemical mode of action. The possibility existed that a functional relationship existed between the two activities or indeed, perhaps, coagulase functioned in some manner with phosphatase action. However the occurrence of another activity in a protein preparation, which has been purified, presents one with the questions of whether the two activities represent the same or different functional groups on the same protein, or on different proteins. 63 When studies on the effect of temperature on the coagulase and phosphatase were carried out no differential effects were revealed. Both activities were inactivated to the same extent after the same times of heating. Since thermal inactivation is usually considered to be responsible for the rupture of chemical bondings through- out a protein structure and not any one specific type of bond, the results suggested that the same or a very similar protein was involved with both activities. To ascertain if the above possibility did, in fact, 5 exist, and that the phosphatase was not simply a separate I molecular entity, co-purified with the plasma clotting activity, numerous attempts were made to bring about separation of the two activities. Anion exchange chromat- ography with DEAE-cellulose proved unsuccessful. At both pH 7.5 and pH 8.6, the coagulase and phosphatase activities emerged simultaneously from the column when subjected to gradient elution with sodium chloride. A series of electrophoretic studies were also conducted to achieve separation. Different buffer systems of various pH values were employed. Insoluble starch, starch gel and paper were used as a stabilizing media. Under the numerous experimental conditions employed, no successful separation of the two activities was obtained. Consequently, the possibility that the coagulase and ph03phatase activities were associated with two different proteins was, indeed, reduced, although not entirely eliminated. However, all 54 one can state with certainty in the light of the experimental results obtained, is that the two activities 'were physically inseparable under the experimental means employed. Since the indication was that one protein was assoc- iated with both the coagulase and phosphatase activities, it remained to be resolved whether these two activities functioned by the same mechanism. The purified prepara- tion was treated with various enzyme inhibitors to deter- mine whether any differential effects were present. Since’ in no case was the degree of inhibition exerted by these compounds on each activity the same, it was concluded that the same functional groups were not involved. Further problems of interest are posed by the studies with the enzyme inhibitors. Contrary to the findings of Drummond and Tager (1959b), it appears from the results that both the phosphatase and clotting activities of the coagulase are dependent upon sulfhydryl groups, the latter activity to a lesser extent. Similarly both activities were inhibited by sodium fluoride and EDTA suggesting the involvement of some essential metal(s). However many extensive studies of the effect of inhibitors on the plasma clotting system must be conducted before any firm conclusions may be drawn concerning the mode of coagulation. Subsequent experiments involving the saturation of the phosphatase activity of the coagulase preparation with 65 excess substrate verified the assumption that the same functional group was not involved with the two activities. Studies were also carried out to determine the increases in specific activity of the staphy10phosphatase and staphy- locoagulase during various stages of the purification process. Since these values paralleled each other, the evidence again indicated that the same protein was associated with both activities. However it should be realized that for proteins of a very similar but not identical structure, it is undetermined whether any of the methods utilized would discriminate between two such compounds. The evidence presented in this thesis represents an attempt to shed some light on the mode of action of staphylocoagulase. The results of our experimentation suggests the hypothesis that the coagulase and phosphatase activities represent two different functional groups associated with the same or a very similar protein entity. However, the author wishes to reemphasize the fact that to prove that this type of phenomenon exists, one must employ data which are usually of a suggestive, rather than conclusive nature. For example, even though numerous electrophoresis and exchange chromatography investigations indicated that the two activities were inseparable, this does not eliminate the possibility that another procedure might yield two separate entities. The absolute resolution of this problem will eventually require precise determination of molecular structure. SUMMARY A purified preparation of coagulase froml§. aureus, phage propagating strain 70, was found to possess a concomitant phosphatase activity. In order to clarify this dual phenomenon a series of studies was undertaken. Thermal inactivation at 37 C and 56 C showed a parallel \ _ ‘77:..1." decrease in both activities. The preparation was subjected to DEAE-cellulose column chromatography at pH v _‘T'T: 1' 7.5 and pH 8.6. Coincident maximum peaks of the two __._ -l—l- _ _._. activities suggested that both functional groups were "ti. 2* associated on the same protein. The two activities, when subjected to starch, starch gel, and paper electro- phoresis under differing conditions of buffers and pH (pH 4.2 to 10), exhibited similar maximum peaks of activity after migration. An extremely small degree of separation may have been obtained when electrophoresis was carried out with a discontinuous buffer system. However, it was not possible to recover either activity in the complete absence of the other, because of the large amount of overlapping of the two activities. EDTA, iodoacetate, fluoride, azide, and p-chloromer- curi-benzoate exerted different rather than similar degrees of inhibition on each of the two activities indicating that they do not function by the same mechanism. This supposition was supported by complete saturation of the phosphatase with excess p-nitro- phenylphosphate substrate (loo-fold K3 value) and the 66 67 demonstration that under this condition the coagulase was not inhibited. Comparable increases were found in the total activity (on the basis of protein) for both the coagulase and phosphatase after each purification step. It is therefore hypothesized that the coagulase and phosphatase represent two different functional groups associated with the same or an extremely similar protein entity. BIBLIOGRAPHY ALDER, V. G., GILLESPIE, W. A., and HERDAN, C. 1953 Production of Opacity in egg-yolk broth by staphy- lococci from various sources. J. Pathol. Bacteriol., 66, 205-210. ALLEN, R. G. L. 1940 The estimation of phosphorous. .Biochem. J., 4, 858-865. BARBER, H., snooxssinx, B. H. L., and KUPEB, s. w. A. 1951 Staphylococcal phosphatase, glucuronidase and sulphatase. J. Pathol. Bacteriol., é}. 57-64. BARBER, M. and KUPER, S. H. 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