An immunochemical study of human plasminogen and plasmin

Immunochem/stry.Pergamon Press 1966. Vol. 3, pp. 29-40. Printed in Great Britain AN IMMUNOCHEMICAL STUDY OF HUMAN PLASMINOGEN AND PLASMIN* C. Rossms and Loum SUMMAmA

Biochemistry Section, Blood Center, Division of Medicine, Michael Reese Hospital and Medical Center, Chicago, Illinois

(Receiwd 28 ~

1965)

Al~tr-..ct~Specific rabbit precipitating antibodies have been prepared to human p!nnfiaogen and phamin. The preoetmyme-entTme pair were found to be indentical in gel diffusion. In immunoelectrophoresis,the proenzyme-enzyme pair ~ in their electr0phore~c mobilities. Antibody y-globulin pre1~nttiOns tO eithe~rhuman phsminogen or plm~nin were found to neutralize the proi~lytic activity of phsmin with waTimum inhibitions of 80 per 'cent. There were some differences in the ability of antibody y-globulin to neutralize plasminogm activated in situ, with either streptokinase or urokinase, as compared to plamnin. The antibod/es also partially inhibit the activation of proenzyme to enzyme. Trypain inldbitora which inhibit plasmin, appear to be different, in certain respects, from specific antibodies.

INTRODUCTION IMMUNOCHEMICALstudies on the relationship of proenzyme to enzyme have been carried out with pepsinogen and pepsin, a-a) chymotrypsinogen A and chymotrypsin A,(4,5) and procarboxypeptidase A and carboxypeptidase A. (o) With the pepsinogen-pepsin and procarboxypeptidase A-carboxypeptidase A pairs, the antiproenzyme reacted in partial identity with the enzyme.(~,e) Whereas with the chymotrypsinogen A-chymotrypsin A pair, the antiproenzyme reacted identically with proenzyme and enzyme.'4) Antibodies to proenzymes and their corresponding enzymes have been found to inhibit enzyme activity(1,4,e) and also to inhibit activation of the proenzyme.a,4,e,7) These antibodies have also been used to detect conformational changes during the conversion of proenzyme to enzyme.(~,a) Antibodies to human plasminogen have been prepared which inhibit proteolytic activity and partially inhibit plasminogen activation. (s) The availability of highly purified human plasminogen and plasminC,,lO) has made possible a more extensive immunoehemical study of this proenzyme--enzyme pair. A preliminary report has been presented. (u) In this study rabbit antisera to human plasminogen and plasmin were tested for their ability to react with proenzyme and enzyme by gel diffusion (Ouchterlony) and immunoelectrophoretic methods. The effect of purified antibody ~,-globulins on the proteolytic activity of plasmin as well as their ability to inhibit plasminogen activation by streptokinase and urokinase was examined. EXPERIMENTAL

Antigens. Human plasminogen and plasmin (streptokinase and urokinaseactivated) were prepared from human plasma Fraction III2,8 by two different methods.(9,x0) Method I included acid and alkali denaturation steps and gave preparations with specific proteolyt/c activities of 20 casein units per mg of * This investigation was support~ by Public Health Service Research Grant No. HE-04366 from the National Heart Institute. 29

30

C. ROBSINSand Lotns SUMMA

protein. ~9~ Method II gave more native preparations with specific proteolytic activities of 24 casein units per mg of protein, c10~ Antisera. l~abbits were immunized with plasminogen and plasmin preparations by a multiple injection technique with the complete Freund adjuvant. The animals were given subcutaneous injections of between 3 and 5 mg of protein at weekly intervals for 3 weeks and were bled at 5, 9 and 13-week intervals after the last injection. The antisera were stored at 4 ° preserved with 0.01 per cent Merthiolate. ~,-Globulin. y-Globulin fractions were isolated from antisera by the Kekwick sodium sulfate precipitation method, os~ The final precipitate was dissolved in 0.067 M phosphate buffer, pH 7.4, and dialysed against several changes of the buffer for 4 days at 4 °. The solutions were clarified by centrifugation and preserved with 0.01 per cent Merthiolate at 4°; they contained 4 + 1 per cent protein. Gel diffusion in agar. Micro-Ouchterlony plates were prepared by a method previously describedCg~ on 3 ×2-in. microscope slides using 1 per cent Ionagar No. 2 (Oxoid) in 0.05 M Veronsl-0.05 M lysine buffer, pH 8.4, containing 0.01 per cent Merthiolate. A single addition of 10 ~1 of antigen containing approximately 20 pg of protein and a single addition of 150/~1 of antiserum was used. Antigen and antibody were allowed to react at 4 ° for 48 hr. The slides were washed, dried and stained with Amido black 10B. Immunoebctrophoresis. A modification of the Scheidegger micromethod a3~ was used as previously described ¢9~ on 3 × 1-in. microscope slides using 1 per cent Ionagar No. 2 in 0.05 M Veronal-0.05 M lysine buffer, pH 8"4, containing 0.01 per cent Merthiolate. Electrophoresis was carried out for 2 hr at 2-3 V/era. A single addition of 2.5 #1 of antigen containing approximately 5 #g of protein and a single addition of 100 tzl of antiserum was used. Antigen and antibody were allowed to react at 4 ° for 48 hr. The slides were washed, dried and stained with Amido black 10B. Quantitative determination of predpitable antibody, a4~ Mixtures of varying amounts of antigen and constant amounts of antibody y-globulin (1"0 mg), in 0"067 M phosphate buffer, pH 7.4, were maintained at 37 ° for 1 hr and then at 2 ° for 48-72 hr. T h e precipitates were removed by centrifugation and washed twice with ice-cold buffered 0.9 per cent NaC1, pH 7.4. They were dissolved in 0.25 N acetic acid and the absorbaney of the solution was measured at 277 m~. Antibody concentrations of y-globulin preparations were calculated from precipitates at equivalence or in slight antigen excess by subtracting the known amount of added antigen (absorbancy at 277 rn#). An extinction coefficient, --1~,~1%of 13.0 was used for rabbit y-globulin to convert antibody absorbancy values to antibody protein. Proteolytic activity. Plasminogen and plasmin proteolytic activity was determined by a modification of the method described by Remmert and Cohn c15~with Sheffield devitamined casein as the substrate.~9,19~ Proteolytic activity is defined in casein units, a-Casein (Borden) which was used for the inhibition studies was found to be a more reactive substrate than ~-casein, K-casein or Sheffield devitaminized casein which is a mixture of the various casein components (Fig. 1). Determination of inhibition of proteolytic activity by antibody. An amount of 1.5+0.5 casein units of either plasmin or plasminogen with activator [500 Christensen units of streptokinase (Varidase--Lederle) or 500 Ploug units of uroklnase (Abbott)] was mixed with 0-0.6 mg of antibody y-globulin, in a volume

Immunochemistry of Human Plasminogen and Plasm/n

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FIC. 1. The hydrolysis of different caseins by 2.0 casein units of plasmin I (streptokinase-activated). of 2 ml containing 0"067 M phosphate buffer, pH 7.4, and incubated for 5 min at 37 °. A volume of 2 ml of ice-cold 4 per cent =-casein was added, the mixture was incubated at 2 ° for 1-3 hr and then assayed (incubation at 37 ° for 30 min--see assay method). The mixtures of plasminogen and activator were pre-incubated for 5 min at 37 ° to form plasmin. In those experiments where activator was added to the mixtures of plasminogen, antibody and casein which had been pre-incubated, it was added immediately before assay. In control experiments, activation of plasminogen by 500 units of streptokinase or 500 units of urokinase was instantaneous in the assay. RESULTS

Immunochemical characteri=ation of plasminogen and plasmin. Plasminogen and plasmin (urokinase- and streptokinase-activated) were found to be immunologically identical in gel diffusion using antisera prepared against these proteins (Fig. 2).~9,10~ These proteins appear to be homogeneous in both gel diffusion and immunoelectrophoresis (Fig. 3) but plasminogen and plasmin differ in their eleetrophoretic mobilities in agar gel. Quantitative determination of plasminogen and plasmin antibodies. The behavior of plasminogen and plasmin in the quantitative precipitin reaction using two antibody ~-globulin preparations is shown in Fig. 4. The antigens behaved similarly with each antibody preparation. The precipitating antibody concentration per mg of ~-globulin in a series of antibody preparations against homologous and heterologous antigens are summarized in Table 1. Plasminogen and plasmin are both good antigens in the rabbit.

32

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C. RoemNsand Loum SUMMAmA

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Fro. 4. Quantitative preeipitin reactiom with antipl~ainogen II-107A and antiplasmin II-117B (urokinase-activated plasmin) and plasminogen II (O,e), streptokinase-activated plasmin II (A,A) and urokinase-activated pl~min II (rn,m). TABLEI.

PRECIPITATING ANTIBODY CONCENTRATIONS OF ANTIBODY ~ - G L O B U L I N PREPARATIONS

Preparation

Antibody y-Globulin

Plasminogen I Plasminogen II Plasmin I (urokinase-activated) Plasmin II (urokinase-activated) Plasmin II (streptokinase-activated) Normal

No. 63AB 107A 65A l17B 1! 9A

Precipitating Antibody Concentrations* (meg antibody/rag y-globulin) 61 4-6 165 4-10 1594-8 604-3 604-8 0

• Three different test antigens were used: Plasminogen II and Plasmin II (urokinase and streptokinase-activated).

Inhibition of proteolytic act/m'ty by ant/body. Plasmin proteolytic activity can be inhibited or neutralized by either phmminogen or plasmin antibodies since the addition of antibody to an enzyme-casein system slowed down the rate of hydrolysis of the substrate (Fig. 5). Antiplasminogen II-107Aneutralized activated plasminogen II and plasmins I and II similarly, giving the same maximum inhibition of

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5 6 7 8 9 10 11 42 FIG. 3. Immunoelectrophoretic patterns of plasminogen II and plasmins II (streptokinase- and urokinase-activated). Antigens: Wells 1, 5 and 9 - plasminogen II; Wells 2, 6 and 10--plasminogen II (1:2 dilution); Wells 3, 7 and 11--urokinase-activated plasmin I I; Wells 4, 8 and 12--streptokinaseactivated plasmin II. Antisera: (a) antiplasminogen II-107A; (b) antiplasmin II-117B (urokinase-activated plasmin); (c) antiplasmin II-119A (streptokinase-activated plasmin).

Immunochemistry of Human l ~ m m m ~

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FIG 5 Inhibition of proteolytic activity of urokinase-activated plasmin I by 0 5 rag antibody F-globulin of antiplasminogen 1-63AB (A) and antiplasmin 1-65A (urokinase-activated plasmin) (m). The antibody was added 10 min after start of assay.

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antiplamfioopn II.107A. Activated plmminosem II: strepmkinue before antibody, v, streptokinase after antibody, y ; ~ - ~ before antibody, @, urokinase after antibody,, e . Umkinase-sctivated ~mm~in I, [3; urokinaseactivated plasmin II, o ; streptokinsse-ac-fivated plasmin II, A.

33

34

C. ROBBINS and Louts SUMMAmA

654-10 per cent (Fig. 6). In these experiments plasminogen was activated to plasmin by streptokinase or urokinase in two different ways; activator was added either before or after enzyme was incubated with antibody. This experimental arrangement allowed a study of the inhibition of activation of plasminogen by antibody. With a second antiplasminogen II-107A preparation, obtained from another rabbit, the maximum inhibition was found to be 384-7 per cent. Antiplasminogen 1-63AB gave entirely different results (Fig. 7). With this antibody

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antiplasminogen 1-63AB. Activated plasminogens II: streptokinase before antibody, v ; streptokinase after antibody, ~' ; urokinase before antibody, ~ ; urokinase after antibody, e. Urokinase-activated plasmin II, o ; streptokinase-activated plasmin II, zx. preparation, plasmin II and streptokinase-activated plasminogen II (activator added after antibody) were neutralized to a greater extent than the other activated plasminogens II. Also, an amount of 0.6 mg of precipitating antibody, in the zone of antibody excess, which neutralized 704-10 per cent of plasmin II and streptokinase-aetivated plasminogen II did not neutralize in any amount the other activated plasminogens. Different types of plasmins can be distinguished by this antibody preparation. Antiplasmin 1-65A (urokinase-activated plasmin I 1 neutralized activated plasminogen II and plasmins I and II giving the same maximum inhibition, 66 + 13 per cent. These data were similar to those obtained with antiplasminogen II-107A. T h e data obtained with antiplasmin II-117F (urokinase-activated plasmin II) were also s'mailar to those obtained with anti. plasminogen II-107A and antiplasmin 1-65A, giving a maximum inhibition ol

Immunochemistry of Human Plasminogen and Plasmin

35

50+ 10 per cent, but, with one exception. Streptokinase-activated plasminogen (activator added before antibody) reacted in the same way with this antibody preparation as with antiplasminogen 1-63AB. The only unusually different antibody preparation was antiplasmin II-119A (streptokinase-activated plasmin II). Several of the activated plasminogens could not be neutralized by this antibody preparation although the plasmins were neutralized about 40 per cent (Fig. 8).

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Fzo. 8. Neutralization of activated plasminogen II and plasmins I and II by antiplasmin II-119A (streptokinase-activated plasmin). Activated phu.ninogens II: streptokinase before antibody, v; streptokinase after antibody, y ; urokinase before antibody, o ; urokinase after antibody, Q. Urokinase-activated plasmin I, []; urokinase-activated plasmin II, o ; streptokinase-activated plasrnin II, 4 .

Antibodies to plasminogens I and II and plasmins I and II generally neutralize plasmin but some of the antisera behave differently in that they will neutralize plasmin and not the activated plasminogens. In most of the experiments maximum inhibition of 1.5+0.5 units of enzyme was obtained with 0-4+0.2 mg antibody. None of these antibodies can differentiate between the urokinase- and streptokinase-activated plasmins. The complexing of streptokinase with plasminogen apparently does not interfere with the interaction of antibody, and inhibition by antibody. None of these antibody preparations could completely neutralize the proteolytic activity of the enzyme. The partial degradation of antibody by plasmin during incubation of the antigen-antibody mixture may be responsible for the inability of the antibody to completely neutralize the enzyme. An analysis of antibody inhibition of the urokinase- and streptokinase-activated plasmins by antibodies to plasminogens I and II and plasmins I and II showed

36

C. Romsn~ and Loum StrmaAmA

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FIG. 9. Inhibition of activation of plasminogen IT by streptokinase added before ( x, [], z~, v, O) or after (+, 1 , 4 , V, e) antibody. Antiplasminogen 1-63AB: O,o; antiphaminogen II-107A: A,A; antiplasmin 1-65A (urokinase-activated plasmin): v,V ; antiplasmin II-119A (streptokinaseactivated plasmin) x, + ; antiplasmin II-117B (urokinase-activated plasmin): D,I. that all antibodies inhibited plasmin but with some variation in degree of inhibition, 60 + 20 per cent. The antibody preparations varied widely in their inhibition of activated plasminogen, whether activator was added before or after addition of antibody. Antibody inhibits the activation of plasminogen by streptokinase since greater inhibition is obtained when antibody is added before activator (Fig. 9). Antibody preparations vary in their ability to inhibit activation of plasminogen. Also, TABLE2. P R E C I P I T A T I N G ANTIBODY C O N C E N ~ T I O N S

OF ANTIBODY T - G L O B U L I N PREPARATIONS D U R I N G I M M U N I Z A T I O N~

Antibody T-Globulin

Preparation Plamninogen II Plasmin II (urokinase-activatexl) Plasmin II (streptokinase-activated)

No.

1434B 140A 149B

PrecipitatingAntibody Concentrations (mcg antibody/rag y-globulin) Bleeding No. I

2

3

50 76 87

91 128 94

26 97 63

• Bleedings 1, 2 and 3 were obtained at 5, 9 and 13-week intervals after last injection.

Immunochemistry of H u m a n Phumfinog~ and Plasmin

TAeLS 3.

37

NBUTRALIZLNG AC'ITVlTY OP ANTtJ~ODY T-GLOBULIN PRI~A]gATIONS DURING IMMUNIT~TION

Antibody y-Globulin Preparation

Plasminogen II Plasm/n II (urokinase-activated)

Inhibition of Plumin II (uroklnase-activated) - Amount of y-Globulin No. Bleeding No. (0.2 mg) (0.4 mg) 1434]] 140A

Plasmin II (streptokinase-activated) 149B

1 2 3 1 2 3 1 2

3

% neutralization % neutralization 30 43 30 34 35 29 43 52 42 71 47 69 45 52 44

46

58

54

smaller amounts of antibody are able to neutralize the same amount of enzyme ff added before activator. The results with urokinase are variable (Figs. 6-8), although smaller amounts of antibody will also neutralize the same amount of enzyme (urokinase-activated) ff added before activator. Plasminogen may also have more antibody binding sites than plasmin. During immunization, the antibody content of the y-globulin preparations after 9 weeks was higher than after 5 or 13 weeks (Table 2). The inhibiting or neutralizing activity of antibody per mg of y-globulin appears to change somewhat during immunization (Table 3; also Table 2). The presence of non-neutralizing precipitating antibodies and non-precipitating neutralizing antibodies in the antibody preparations is also possible. Since all antibody preparations neutralize plasmin II (urokinase and streptokinase-activated) in the same manner, the neutralizing antibody is probably the precipitating antibody. The neutralizing ability of antibody was maximum when the antigen-antibody-substrate mixture was assayed within 3 hr after mixing at 2 °. After 24 hr, the antigen-antibody complex, in the presence of substrate will partially dissociate resulting in less inhibition. The normal rabbit y-globulin preparations used as controls in the experiments contained no precipitable antibody but several preparations were found to contain some neutralizing antibody. The F-globulin fractionation procedure should have eliminated normal serum antiplasmin activity but some small amount may have been concentrated in the y-globulin preparations. The maximum inhibition found with several normal rabbit ~-globulin preparations was 22 per cent but most of the preparations gave no inhibition. Several of the normal rabbit F-globulin preparations contained small amounts of plasminogen (streptokinase-activatable). The interpretation of the results obtained with antibody concentrations of more than 0.5 mg antibody are complicated by the presence of increased proteolytic activity in the control experiments. Greater inhibition may have been found at higher levels of antibody ff plasminogen were absent. Antiplasminogen II-107A will inhibit the hydrolysis of specific phsmin synthetic substrates, e.g. lysine methyl ester, lysine ethyl ester and tosyl arginine methyl ester, all to the same extent, with maximum inhibition of about 55 per cent.

38

KZNNgrH C, ROBmNS and Loum SUMMARIA

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80 70 o~" 6 0 t-

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FIG. 10. Inhibition of urokinase- ( o , o , ~ , v ) and streptokinase-activated ( e , I , A , V ) plasmin II by pancreatic (o,o), soybean (D,I), and lima bean (A ,A) trypsin inhibitors and ovomucoid (v ,T).

Effect of trypsin inhibitors on neutralization of enzyme by antibody. Certain trypsin inhibitors, e.g. pancreatic, soybean and lima bean, will inhibit both streptokinaseand urokinase-activated plasmin II (Fig. 10).(16,17) Ovomucoid, which does not inhibit plasmin significantly, will differentiate between plasmin and trypsin. These inhibitors were used to study the antibody binding site. When a pre-incubated mixture of enzyme (II) and antiplasminogen II (50 per cent inhibition) was mixed with trypsin inhibitor (50 per cent inhibition) and casein, it was found that with urokinase-activated plasmin II, the maximum inhibition was 88 per cent for pancreatic and soybean inhibitors and 76 per cent for lima bean inhibitor. But with streptokinase-activated plasmin II, the maximum inhibition was the same as the control enzyme-antibody mixture without inhibitor. The addition of inhibitor to enzyme and antibody, did not give any additional inhibition of streptokinaseactivated plasmin II. But, urokinase-activated plasmin II, in this system, was inhibited an additional 26-38 per cent. There are apparent differences ,in the enzyme binding sites between streptokinase-activated plamfin and urokinaseactivated plasmin. Also, with streptokina~-activated plasmln the binding sites of antibody and inhibitor appear to be either the same or in close proximity since the results are similar even when inhibitor was added to enzyme before antibody. In these latter systems, once inhibitor (50 per cent inblbition) or antibody (50 per cent inhibition) is bound to enzyme, no further inhibition was found by the further addition of more antibody or inhibitor, respectively. Since all antibodies do not

Immunochemistry of Human Plasminogen and Plasmin

39

completely inhibit plasmin, the antibody binding site is probably not the active site of the enzyme. Also, since the trypsin inhibitors can completely inhibit plasmin, the antibody binding site is probably different from the inhibitor binding site.

DISCUSSION Highly purified human plasminogen and plasmin which were immunochemieally homogeneous showed reactions of identity with specific rabbit antibodies (Fig. 2).~9,10~ This system is another example of a proenzyme-enzyme pair reacting with identity with antibody to either proenzyme or enzyme. This reaction was similar to the reaction of the chymotrypsinogen A--chymotrypsin A pair with antibody~4~ but different from the reaction of the proearboxypeptidase A-carboxypeptidase A pair and the pepsinogen-pepsin pair with antibody.*2,0~ In immunoelectrophoresis, the positions of the plasminogen and plasmin-antibody bands were found to be different (Fig. 3) indicating different electrophoretie mobilities for plasminogen and plasmin. Similar immunoelectrophoretie results were found with the proearboxypeptidase A-carboxypeptidase A pair. ~e~ The two systems which showed reactions of identity showed no gross changes in molecular weight after activation.a0,ls~ But in those systems showing reactions of partial identity, large changes in molecular weight were found.a°,~°~ In the conversion of human plasminogen to plasmin, the sedimentation and diffusion coefficients increased but the molecular weight remained the same, namely, 89,000.a0~ Also, the frictional coefficient changed from 1.8 to 1.5 indicating a change toward a more compact molecule.~0~ These eortformational changes did not influence the antigenic properties of the molecule (Figs. 2 and 3). The eonforrnational change may be a result of the liberation of a small peptide during the activation process.~21~ Also, plasmin is more susceptible to reducing agents such as 1 M mereaptoethanol, in 8 M urea, resulting in the formation of subunits.~l~ Antibodies to proenzymes and enzymes have been found to inhibit enzyme activity and to inhibit activation of proenzyme.a,v~ The inhibition of plasmin proteolyti¢ and fibrinolytie activity by rabbit precipitating antibodies to human plasminogen was previously reported. ~s~ The human plasminogen used in these studies ~s~ was a crude and immunologically heterogeneous plasminogen I type of preparation. The rabbit antibody preparations used were purified y-globulins; the experiments were carried out with streptokinase-activated plasminogen as the enzyme and casein as the substrate. The rabbit precipitating antibody y-globulin to human plasminogen was found to inhibit plasmin activity and to partially inhibit plasminogen activation. These findings were confirmed in this report. Specific precipitating antibodies to human plasminogen and plasmin are neutralizing antibodies. The antibody preparations behaved differently in their ability to neutralize different types of plasmins. The partial inhibition of plasminogen activation (by streptokinase) by antibody (Fig. 9) may be due either to inhibition of activation or to the inability of the activator to reach the proenz3,me active center, perhaps due to sterie hindrance. Antibody was also found to inhibit the formation of bovine plasminogen activator.~m~

40

C. Ronn,~s and Louis SUMMaaLa

It has been suggested that activation of proenzyme to enzyme takes place during immunization resulting in antibodies only for the enzyme.CS~This possibility exists for human plastrdnogen since most tissues contain activators of this proenzyme. But, inhibitors of activation and antiplasmins are also found in most animal tissues and blood. Ackno~ledgement--We acknowledge the technical assistance of Mrs Susan Lu and we are indebted to Dr C. Ling for his comments and criticisms. REFERENCES 1 VAN VUNAKISH., Lmmza H. I., ALLISONW. S. and LEVINE L., .7. gen. Physiol. 46, 589 (1963). 2 Scw_.a~own'z M., V ~ A N z P. T. and WmSLe~ F. C., Biochemistry 2, 238 (1963). s Scm..AMowrrz M., S H A W A. and JACKSO~ W . T., Biochemistry 3, 636 (1964). 4B ~ J. T. and T H O M ~ O ~ L. D., Immuno/ogy 8, 136 (1965). s Rxcxo.xE. and Ca~n~B~LL D., Fedn Proc. Fedn Am. Socs exp. Biol. 22, 555 (1963). 6 Bam~'x'r J. T., Innnuno/ogy 8, 129 (1965). 7 A ~ o N R. and ~ G. E., 3. bioL Chem. 238, 963 (1963). s M m , m ~ W., BUSDON K. L. and RILEY M. N., Ex"per~tia 14, 280 (1958). ' ROBmNS K. C. and SUMMASlA L., J. b/o/.Chem. 238, 952 (1963). 10 ROBBINSK. C., SUMMAmAL., ELWYN D. and B~mLOWG. H., J. bioL Chem. 240, 541 (1965). 11 ROSBL~SK. C., Lu S. and SUMMA~A L., Fedn Proc. Fedn Am. Sots exp. Biol. 22, 561 (1963). 12 Km~ICK R. A., Biochem..y. 34,1248 (1940). is SCHmDZGG~aJ. J., Int. AreAs Allergy a~l. Immun. 7, 103 (1955). 14 KABATE. A. and MAY~a M. M., Ez.perimental Im~nochemiatry 2nd Ed., p. 22, Thomas, Springfield, Illinois (1961). is ~ T L. F. and COH~ P. P., j~. b/o/. Chem. 181, 431 (1949). is DAvlz E. W. and RATNOFUO. D., In The Proteins (Edited by NEUZATHH.) Vol. 3, p. 359, Academic Press, New York (1965). 17 LASKOWSKIM. and LASKOWSKIM., JR., In Advances In Protein Chemistry (Edited by AusoN M. L., BAIImYK. and EDSALL J. T.) Vol. 9, p. 203, Academic Press, New York (1954). is DZSNU~LE P., In The Enzymes (Edited by BoY~ P. D., LARDY H. and MYRBACKK.) Vol. 4, p. 93, Academic Press, New York (1961). 19 BovEY F. A. and YANARIS. S., In The Enzymes (Edited by BoYlm P. D., LARDYH. and MYRBACKK.) Vol. 4, p. 63, AcademicPress, New York (1961). 90 N~]aATH H., In The Enzymes (Edited by BoYEa P. D., LAZDY H. and MYRSACK K.) Vol. 4, p. 11, Academic Press, New York (1961). Sl ROBBINSK. C., SLrMMARIAL , HSIEH B. arid LING C., Thromb. Diath. Haemorrh. 8, 586 (1965). n LING C., SUMMARXAL. and ROBBmS K. C., to be published.