ON THE ISOLATIQN, PURIFICATION TEREIATIGN 0? RASMINQGEN LASMA FRACTION III STUDIES AND CRARAE FROM HU MAN P of DE. DI Thesns I‘or the Daqmc I‘I’EESETY MICHIGAN STEE {III Gerda Mootse 1959 Am.) an. .uualri l-lm‘: L I II R A R Y M i-C’f. 5 "3‘?! State Umvcrsity IIIIIIII l I IIIIIIIIIIIIIIIIIIIIIII 3193009911110 62451 3.5" I.» .. \ f) ( fr 3 I-I-cr Sm; STUDIES ON THE ISOLAIION, 'URIFICATION ATE CHARACTERIZATION OF PLASfiIUOGEK FROM HUMAN PLASMA FRACTION III By Gerda mootse A THESIS Submitted to the School of Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 1959 7, 157245 6/145” ACKNOWLEDGMENTS The author wishes to express sincere appreciation to Dr. Hans A. Lillevik for his patience, encouragement, interest and guidance during the course of this study. Acknowledgment is also due to members of the Bi010gic Products Section, Division of Laboratories, Michigan Department of Health for providing materials, facilities and funds in support of this work?;.Also appreciation is expressed for advice, helpful criticism, and technical assistance given by members of this group. A portion of this study was also supported in part by a grant from the American Dairy Association. 9Q'This work was done at the request of the Director of the American National Red Cross Blood Program under an agreement between the American National Red Cross and the Michigan Department of Health Laboratories. b— STUDIES ON THE ISOLATION, PURIFICATION AID CHARACTERIZATION OF PLASMINOGEN FROM HUMAH PLASHA FRACTION III By Gerda Mootse AN ABSTRACT Submitted to the SChool of Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 1959 ‘ 7 , ; 24/ x . Approved __C7 j -e fif/L ABSTRACT Purification of the plasma enzymes, enzyme activators, and enzyme inhibitors should give material with which many fundamental Questions can be answered. Therefore, methods that give only reliable and re. peatable preparations have practical significance. Such was the object of this study. The starting material used was Cohn's Fraction III obtained from human plasma which contains two known precursors of proteolytic enzymes, namely, plasminogen and prothrombin. An extensive purification study prior to applying the method of Kline was carried out on Fraction III. Thereafter an improved modifica- tion of the Kline procedure was developed. The initial purification consisted of the elimination of the two main components from the Fraction III, fibrinogen and lipid material. Fibrinogen was first extracted into phosphate buffer at pH 6.“,ionic strength = 0.05. Plasminogen was next dissolved from the remaining ‘precipitate into 0.1 M acetate buffer of pH 4.b, leaving lipid material with the residue. Adjusting the solution to pH 7.h precipitated plas- Ininogen. This precipitate was now ready for final purification by a rhodified Kline procedure. The principal change in the Kline method developed in this study ‘was the fractionation of Solution A (modified) by ammonium sulfate. {Phe precipitate obtained at 0.20 - 0.3M saturation gave the highest ac- tisvity yet reported, its. lZO—lho P.U./mg.N with 30 - 40 per cent yield. The high purity plasminogen appeared homogeneous according to ultra- Centrifuge sedimentation patterns, but various components showed up in electrOphoretic patterns obtained by runs in glycine buffer at pH 2.1. Caseinolytic, fibrinolytic and p.toluene-sulfonyl-L-arginine methyl ester esterolytic activity by the plasminogen preparation was found to be inhibited by cysteine. Lplysine ethyl and methyl ester activities were not inhibited by this agent. The presumed importance of the ~S-S- linkage in the plasminogen molecule is indicated. Cysteine inhibition is suggested to be the re— sult of reduction of -S-S- bond(s) or a disulfide interchange reaction in presence of thiol compounds. I. II. III. IV. V. INTRODUCTION . . HISTORICAL . . . EXPERIMENTAL . . A. Apparatus . TABLE OF CONTENTS B. Materials and Reagents . C. Experimental Procedures . . . Preparative procedures for Plasminogen . . . . Plasminogen activity on TAMe and LEe Hydrolysis The fibrinolytic activity of Plasminogen . . Physics-Chemical prOperties of the highly active Plasminogen. . . . DISCUSSION . WIflmY o o 0 LIST OF REFERENCES . . . . . . . Page 12 12 13 {B ‘83 79 81 Table I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. .XIV. LIST OF TABLES Page Effect ofigPlasminogen Dilution on Proteolytic Activity . . . . . . . . . 19 Liberation of Acidic Groups During Proteolysis of T5 Casein Solution, Using 88 P.U./mg.N Plasminogen, as Determined by Titration in Alcohol and later . 25 Proteolytic Activity and Nitrogen in Various Samples of Fraction III Suspended in 0.008 M Acetate pH 5.“ 29 Proteolytic Activity and Nitrogen in the Supernatant from.Fraction III Suspension . . . 31 Proteolytic Activity and Nitrogen in Supernatants of Fraction III-3 after Treatments Affecting the Clot Formation of Method 9 . . . . . . . . 31 Proteolytic Activity and Nitrogen in Citrate Buffer Suspensions of Fraction III . . . . . 33 Proteolytic.Activity and Nitrogen in the Supernatants from Fraction III Suspensions using Citrate Buffers 3N Proteolytic.Activity and Nitrogen in the Supernatants from the Second Precipitate of Scheme 2 . . . . . . 35 Proteolytic Activity per mg.Nitrogen in Supernatant 0-2 of Scheme 2 as Influenced by pH, Salt and Precipitation Time . . . . . . . . . . 35 Proteolytic Activity and Nitrogen of Supernatants and Precipitates in Step 2 and 3 of Scheme 3. . . . 37 Proteolytic Activity and Nitrogen of Supernatants and Precipitates in Steps 2, 3 and N of Scheme h... 39 Proteolytic Activity (of one sample) and Nitrogen of Supernatants in Steps 2 and 3 of Scheme 5 . . . NO Proteolytic Activity and Nitrogen in Supernatants of Steps 1 and 2 of Scheme 6 . . . . . . . . . . . ho Proteolytic Activity, Nitrogen, Total and Inorganic Phosphorus and Cholesterol in Butanol-Acetone (6 :h) Extract of Lyophilized Paste 02-2 of Scheme 2. ... N2 Proteolytic Activity and Nitrogen of LyOphilized Paste 02-2 of Scheme 2 (lot #1711) after Treatment by Various Steps of Kline Method . . . . . . MM ryove “1.... XIX. III. 1111.11 nIII.‘ XXIV.‘ Table XVI. XVII. XVIII. XIX. XXI. XXII. XXIII. XXIV. XXVI. XXVII. XXVIII. XXIX. Page Proteolytic Activity and Nitrogen of O. 05 N H 230A Extracts of the Last Precipitate of Scheme 3 Through 6 . . . . . . . . . . . . . . . . . . . “5 Proteolytic Activity and Nitrogen of 02-2 Scheme 2 in Various Steps of Kline Method .. . . . . . . . . Mb Proteolytic Activity and Nitrogen Remaining in Super B in the last step of the Kline Method . . . . . . M7 Proteolytic Activity and Nitrogen of the Extract of LyOphilized Paste Fraction III-3 from Scheme 1 life tile d 9 C O O O O O O O O O O O O I O O O I O O O u? Proteolytic Activity and NitrOgen in Precipitates and Supernatants of Fraction III Carried Through the Procedure of Scheme 2 and the Kline Purification Method......................118 Proteolytic Activity and Nitrogen from Four Differ- ent Starting Materials (Fraction III) According to Scheme 2 and the Kline Method (Scheme 7) . . . . . M9 Proteolytic Activity and Nitrogen in Buffer Extracts of Lyophilized Paste 02-2 Scheme 2 . . . . . . . . 50 Partition of Proteins with Saturated (NE A)? PM Acid Extract of AM—Z Scheme 5 . . . . . . 51 Proteolytic Activity and Nitrogen in Supernatant of Modified Kline Method at pH 5.3 . . . . . . . . . . 51 Proteolytic Activity and Nitrogen in Solution A of Modified Kline Method . . . . . . . . . . . . . . . 52 Proteolytic Activity and Nitrogen in Precipitates B and Supernatants B in Modified Kline Method . . . . 52 Specific Activities of Solutions A and of tie (NFhLSO Fractions of them . . . . . . . . . . . . . . . 3 Specific Activities of Precipitates B and (NHh)280u Fractions of them . . . . . . . . . . . . . . . . 5 Proteolytic Activity in Various (Nflu)280u Fractions Precipitated From Acetate Buffer . . . . . . . . . 55 The Yield and Purity of Precipitate B Dissolved in V A-cetate Buffer PH “.6 C O O I 0 O O O o O 0 O 0 0 0 Sb Table Table XXXI. XXIII. XXXIII. XXXIV. XXXV. Page Specific Activity and Recovery of Activity in Acetate Buffer Near the Neutrality . . . . . . . . 57 Plasmin Activity on TAMe and LEe . . . . . . . . . 59 Approximate Sedimentation Coefficients of Plasmin— an at 0. 5° - 1. 5° C in Glycine Buffer pH 2.1; r2=0005 o o o o e o o o a o o o o o o . 61 The Activation Behavior of Fraction III (lot #1711) in 01 trate Buffer pH 6. u '72 = o. 3 During Various Intervals of Time at Room Temperature . . . . . . . on The Inhibitory Effect of stteine and Calcium Chloride . . . . . . . . . . . . . . . . . . . . . 67 Figure 1. 2. LIST OF FIGURES Page The Effect of Enzyme Concentration on Proteolytic Activ ty According to the Procedure of Remnert and COhen O O O O 0 0 O c I 0 O O O O O O O O O O O O 0 Effect OE Streptokinase Concentration on Proteolytic Activity 8 . . . . . . . . . . . . . . . . . . . . . Consumption of Base in Alcohol and Water Media, Dur- ing Digestion of “% Casein Solution . . . . . . . . .. Electrophoretic Patterns of N% Casein at 0 Time and after 30 Minute Digestion . . . . . . . . . . . . . . Spontaneous and 5.x. Activated TAMe Esterase Activity of Plasminogen . . . . . . . . . . . . . . . . . . . Ultracentrifuge Patterns of Plasminogen, In Glycine Buffer pH 2.1; :72 = 0.05, at o.5° - 1.50 c and 59,780 r.p.m. . . . . . . . . . . . . . . . . . . . Electrophoretic Patterns of PlasminOgen . . . . . . . 62 63 INTRODUCTION Recognition of the physiological significance of the fibrinolytic system during thrombotic conditions has promoted.a growing interest in the problems concerning the proteolytic enzymes in human blood. There have been many observations on the similarity of the mech~ anisms of clotting and fibrinolysis, both having parallel systems of plasma and tissue factors. It is possible that recent advances in the identification of clotting factors may reveal links between these systems. As is usual, the extensive work done has revealed a greater comp plexity than was suspected. Factors separated in the laboratory may in life form part of a single complex, the activity of which depends on its integrity. Mammalian plasma proteolytic enzyme system is comprised of many indi vi dual components. Plasminogen, the precursor of the fibrinolytic enzyme found in the blood and active at neutral pH, can be converted to its active enzyme, plasmin, by a variety of activators. The means by which active enzyme is released from the precursor in the blood and the mechanism of this activation, remain obscure. Inconsistent behavior of precursor samples during fractionation Procedures, suggesting coprecipitation with other proteins, has in general resulted in distribution of the proenzyme into every fraction separated, and too frequently, in proportions similar to those in which total protein was distributed. Changes in apparent solubility by factors of several fold have 2 been encountered frequently under supposedly fixed conditions, but at different stages of purification. The principal objects of this study were to discover methods for further purifying plasminogen and to elucidate the properties of both the active and inactive forms of the enzyme. In the following experiments to be described, attempts have been made to study the purification of plasminogen in a more or less system. atic manner step by step using Cbhn's Fraction III from human plasma, as the starting material. HISTORICAL That mamallian blood contains a proteolytic enzyme which causes clotted blood to redissolve was first reported by Dastre, 18931. Reviews of the subsequent literature pertaining to the proteolytic enzyme system have been published by Christenseng, by Kaplan3, by Rocha E. Silva and co-workersu, by Mac Farlane and Biggss, and in 1956 by.Astrup6. The historical background reveals that the development of fibrino— lytic activity in the organimm is the result of a very complicated pro. cess. Experimental findings and conclusions related to fibrinolysis may be summarized in an activation scheme as presented by Astrup in 195k. Plasminogen 4%: Plasmin T(activation of plasminogen) Spontaneous Chloroform Activators of Trypsin Plasminogen Tissue activator Activator in urine (Urokinase) Activator in blood, milk, etc. 4\ Spontaneous Lysokinases Streptokinase , (activation of proactivator) Kinase in blood Tissue kinase Proactivator in blood, milk, etc. Spontaneous activation in vitro may occur during the process of plasma fractionation7, after the removal of a plasma inhibitor by chlorofonm ’ , or under conditions where stabilization of newly formed plasmin has been assured8 . u 8 An investigation by AlkJaersig and co-workers of the spontaneous 9 activation of plasminogen prepared by the Kline procedure has shown that the early methods described were unreliable and unsatisfactory, since both the enzyme and its precursor were unstable. They found that 50 per cent glycerol stabilized plasminogen and plasmin, and under de- fined conditions also induced the complete conversion of plasminOgen to plasmin which remained stable. The appearance of trichloroacetic acid (TCA), soluble moieties in the activation mixture indicated that proteolysis was involved in the activation. The kinetics of the activation of plasminogen by trypsin, strepto- kinase (S.K.) and urokinase zhas been measured by the same authorslo. Their results showed that the activators exerted their effect through an enzymatic process, since Lineweaver-Burk plotsle were linear in each case. During the activation process there was a release of a TCA-sol- uble moiety, equivalent in each case to some 25 per cent of the original TCA-precipitable material. Concerning the role of fibrinolysis in several physiological and pathological conditions one may refer to the article by Hallertzl3. Preparations containing plasminogen or plasmin are very often contamdnp ated with an activator or with its precursor (proactivator) of plasmin— ogen. Hanan blood contains large amounts of proactivator and relatively small amounts of plasminogen,by addition of streptokinase (S.K., bac- terial product of hemolytic streptococci) large amounts of activator and small amounts of plasmin are formedJBOVine blood has much plasmin- ogen, and little or no proactivator. Bovine plasmin is not formed with 1M S.K. . Activation of plasminogen.from various animal species by S.K. iv w" nature-s I83 “I the gr» I plssniJ result C? of pro of the inhibi direct the pr. also be Th synthet ester ( affinit fOre t1. 80 that casein T The metrical J 1 Rob 'ith LEeJ Restrini 3&st on mine J 1 5 was studied by Takayoshi15 with the results that dog and human responded the greatest, thus showing species difference. There exist various procedures for estimating the activity of plasmin. The choice of the method has a profound influence upon the results obtained. Casein, or any other plasminogen-free substrate gives an expression of proteolytic activity (usually evaluated by measuring the absorbency of the deproteinized filtrate at 280 my). In less pure preparations inhibitory agents are usually present and the results will not give a direct estimate of plasmin. The casein method is rather sensitive to the presence of inhibitory agents. The activators of plasminqgen can also be determined by their effect on purified plasminogen with.casein;u. The lytic effect of less pure plasmin can be followed on certain synthetic amino acid esters such as p—toluenesulfonylarginine methyl- ester (TAMe) and lysine methyl- or ethyl-ester (Lie or LEe). The affinity of plasmin for these synthetic substrates is very high. There- fore this estimation is only slightly influenced by inhibitory agents, so that less pure plasmin preparations can be assayed compared to the casein methods. The esterase activity of plasmin has been determined: (a) titri- 16,17 18 metrically , (b) manometrically , (c) determining the free lysine 1Tonmed turbidimetrically in acetonelg, and colorimetrically ' . Roberts in 1958 reported the poor reproducibility (specifically ‘Eith.LEe substrate) by the titrimetric method and suggested a modified Hestrin'seo colorimetric method for esterase activityal. The method is based on the formation of a ferric complex of the hydroxamic acid re- sulting from the reaction of an ester with alkaline hydroxylamine. 6 The tendency of fibrinogen and fibrin to retain large amounts of plasminogen causes fibrinolytic and fibrinogenolytic methods to be essentially a means of estimating the activators of plasminogen. Only when the absence of activating agents in the test solution has been secured can the results be read in tgrms of proteolytic activity with respect to plasmin present. This phenomenon explains the discrepancies encountered in estimation of lytic activity when results obtained on fibrin or fibrinogen are compared with those obtained on casein. A great amount of work has been done to prepare fibrinogen (and fibrin) free from plasminogen for the estimation of fibrinolytic enzymes in the presence of activators of plasminogen. Lassen22 found that the fibrin layer in the fibrin plate method of assay23 could be heated for 30 - N5 minutes at 80° C. without ser- iously interfering with the structure of theklot. This heat—denatured fibrin contained no plasminogen, and inhibitory agents had also been destroyed. This method, the so called heated fibrinpplate method, has been very useful in differentiating between activating agents and pro- teolytic enzyme activity. Factors influencing susceptibility of fibrin and fibrinogen to proteolysis by plasmin has been discussed by Calendar et ale“. Methods for the detection and measurement of human fibrinolysis have been evaluated by V. Kaulla and Schultzes. They could not find any correlation between esterase activity and fibrinolytic activity when the activation of human-plasma plasminogen was brought about by human activator, such as urokinase. This is contrary to the results 16 obtained after activation of plasminogen by S.K. . Other methods of activity determination, used by previous workers inclu Stmt gaard and P plasmi the pier ” by urd 'allén maxima by th: appear O‘J Very (till ent fro 7 include change in viscometric measurement on casein or gelatin 6 sub— strate solutions. Protaminase activity of plasmin has been demonstrated by Kjeld— gaard and Plow;19 on the heparin-protamine complex and by Brunfeldt and Poulsen27 on insulin-protamine complex. Limited proteolysis by '3 . plasmin on corticotr0pin A has been shown by White“’. Plasmin effects the non-Newtonian viscosity and the character of the mucin clot of solution containing the hyaluronic-acid-protein com- 1:191:29 (similar to papain). N-terminal amino acids formed during digestion of bovine fibrinogen by urokinase activated plasmin were determined by Iallc'm et a130, Vania and Bergstrom showed, that plasmin split at least 150 bonds at l maximal fibrinogen digestion3 . The same peptide bonds were attacked by thrombin whether it was digested before by plasmin or not. Upon digestion of pure fibrin and fibrinogen by plasmin, there appeared antithrombin activity in the digestion mixture32. Similarly it has been found that the mechanism of inhibition is very complicated. Inhibitory systems consist of antiplasmin (differ- ent from antitrypsin) and inhibitors against activators and lysokinases. Heat labile trypsin inhibitor in plasma and serum is probably responsible also for a large part of the plasmin inhibiting effect of blood. Jacobsson33 separated by electrophoresis two different trypsin inhibitors. One inhibitor migrated with the 09-1 globulins, and the other with 1-2 globulins. Alpha—2 inhibitor had a potent effect on S.K. activated human plasminogen tested on fibrin. The Oh-l fraction 1; '88 separated by the anion exchange method as described by M011 et a1.3 and it failed to inhibit plasmin. 8 Norman” reported the “-1 and d-Z anti-plasmins of human plasma. The «-2 inhibitor dissociated readily from the plasmin-inhibitor com— plex and only 50 per cent was inactivated after 90 minutes at 60° C. The 4-1 inhibitor did not dissociate readily and was destroyed at 50° c for 30 minutes. Both inhibitors were inactivated at 25° C at a pH below 5.5 or above 11.0. A proteolytic inhibitor of plasmin with anticoagulant activity has been separated from human urine and from isoelectrically precipi ta— ted serum globulin by Shulman36. The reaction of the inhibitor with plasma proteolytic enzymes was reversible. The inhibitory effects of a number of inorganic and organic com— pounds, including dyes, have been studied. Metallic Ions. Copper and zinc ions in 0.01 H final concentrations completely inhibited spontaneous activation in glycerol . Calcium, magnesium and manganese had no such effect . Calcium inhibition of active fibrinolytic enzyme in normal plasma has been shown by Fearnlyn to be maximal at the lowest plasma dilution. Thromboelastographic studies of S.K.-induced fibrinolysis by O'Neal and 38 Tillman showed the same inhibitory effect by Game. Ratnoff39 reported rapid activation of precursor (plasminogen) by calcium ions in human Plasma. However, partially purified plasminogen was not activated by calcium ions. As the investigations presented by various authors are still of a rather preliminary nature, there are controversial statements concom- ing the effect of various ions and various organic compounds. Alstrup6 found the activation or inhibition effect of heparin to depend upon pH and ionic strength of the fibrin substrate and on the 9 plasmin sample. Therefore it appears possible to bring into harmony previous discordant observations about heparin. Plasminogen is unaffected by diisopropylfluorophosphate (DFP), but plasmin was inhibited non-competitively . Plasmin inhibition by toxic phosphorus compounds has been studied by Mounter and Shipleyuo. Such protein combining dyes as congo-red, u trypan-red, trypan-blue did not inhibit fibrinolysis 1. 1&2 Ambrus et al. reported severe allergic reactions of living organ.- 5 sms in response to injection of S.K. or S.K. activated plasmin. How- ever, no anti-bodies were produced upon introduction of urokinase or urokinase activated plasmin. A report by Ferguson et all?3 ascribes a weak trypsin-like thrombo— plastic activity to the fibrinolytic enzyme present in normal human blood, suggesting participation of this proteolytic enzyme in blood coagulation. This observation was questioned by Seegers et alm. The findings reported by V. Kaullaus in 1958 miss anew the question of the interaction of fibrinolytic enzyme and clotting factor. It was observed that induction of in m fibrinolysis in human blood either by S.K. or bovine fibrinolysin was always precedod by earlier onset of fibrin formtion. Plasminogen. Preparations containing plasminogen have been usually Purified by isoelectric precipitation or by ammonium sulfate fraction.- at1 on of blood plasma or serum. Partial purification of the enzyme was reported by Edsall et allfé, Loomis et a197, Rocha E. Silva et al., {‘8 . and Remert et a1 . The principal obstacle to purification has been the intense co-precipitating properties of plasminogen. For this rFifi-Son, the classical techniques of salt, alcohol, and isoelectric fractionation have proved of limited value. 10 The extreme stability of plasminogen toward acid was used for basis of purification by Christensenug. 'I'agnon‘j0 in 1914.? showed that the activity of the fibrinolytic en- zyme was connected with the euglobulin fraction of the blood. In 1951 Christensen and Smith“9 were the first investigators to utilize the 1 Cohn Fraction 1115 as a starting material for plasminogen of human origin. Plasminogen is one of the least soluble of plasma proteins, being found in the residues after extracting the other components of Cohn's Fractions I and II + 1117. Kline9 by using Christensen's acid extraction in connection with selectively denaturing the interacting contaminants at high pH, ob. tained the concentration of enzyme more than l#00 times that of serum. Few attempts have been made to purify the activated enzyme. In 1956 Kline and F‘isl'lnan‘j2 reported 2.5 times purification over that of high purity plasminogen by fractionating the active enzyme with alcohol. Physico-chemical properties of plasminogen and plasmin Inve been in- vestigated by Shulman and co-worker853, and some of their data may be summari zed as follows: 53 A Summary of Properties ¥ Plasminogenfliunnn) Plasmin( Human) Casein units per mg. of Na 60-100 Molecular Weight 1143.000 108,000( spontaneous) 5.6 6.2 I soelectric point Electrophoretic mobilities in 7 5 2 glorcine buffer pH 2.1 8.2x10'gcmgper v per sec. 8.91:10" cm per v per 7.3x10- cm. per v per sec. sec.(spontaneous) Tryosine 5.91% 6.3 Tryptophan 3 . 78% 14 .014 Ratio Tryosine to Tryptophane 1.56 1.56 N1 trogen 15.8 fi 114.]. ‘X Phosphorus 0.19% Hexose 0.98% 1.51% a)Proteolytic or caseinolytic unit--1 proteolytic unit was arbi- trarElly taken as the amount of enzyme producing an increase of (of acid— soluble tyrosine in a medium of 14 per cent casein in 1 hour . 11 Ultracentrifugal information about changes during activation of human plasminogen has been reported by Harms et alsu. All of these consti- tutional data are at present open to some degree of uncertainty because of the heterogeneity of the preparations. For instance, the physica- chemical data of plasminogen by Reitzenhoffer et al5.5, who reported 70 per cent purity as determined electrophoretically, does not compare with Shulman's data. Enzymatic properties of bovine plasmin have been characterized by Ronwins6 and compared with those of thrombin and trypsin57. He empha. sized an extraordinary similarity: witness their action on the same substrates, identical pH optimum curves, etc. However, plasmin and trypsin showed marked resistance to combined acid and heat treatment 56 while thrombin rapidly deteriorated under the same conditions . EXPERIMENTAL A. Apparatus: Temperature control—- A constant temperature bath sold by the Amer- ican Instrument Co. and equipped with a Thryratron relay Aminco thermo— regulator unit which controlled the temperature at 37.50 t 0.050 C was used. For low temperature control, constant low temperature thermostat tanks and refrigerated centrifuges controlled to any required tempera- ture were employed. (The Servall refrigerated centrifuge with size SS-l rotor for 50 ml tubes and International Refr igerated centrifuge Model FRp1.were utilized.) pg 233357. A Beckman Model G or Model HZ, line operated pH meter was used for hydrogen ion activity measurements. Stability for the Model H2 was improved by use of a 115 volt Sola constant-voltage trans- former between the line and the instrument. Values of pH were deter~ mined with glass electrode. As reference electrode the calomel cell was used, and for calibration at pH 7.0 Beckman standard buffer was properly diluted or at pH n.0, 0.0500 M acid phthalate was employed. Timer-— A Meylan stop watch was used to time the reaction periods. Spectrophotometers-- Absorbancy measurements at 280 mu were made using the Beckman Model DU spectrOphotometer. The Beckman Model B spectrophotometer was used in the determination of Biuret nitrogen and for esterase activity according to modified Hestrin's colorimetric method. Dialysis-- All the dialyses were made in Visking cellophane tubing using an external rotating liquied dialyzer constructed according to 13 DJang, W58 . ElectrOphoretic Analyses-- were made with the Tiselius electrophor- esis apparatus Model 138 (Perkin Elmer Corp.). For conductivity mea— surements, the Model RC—IB resistance bridge (Industrial Instruments Inc.) attached to a conductivity cell (Perkin Elmer) of cell constant = 0.1893 was used. Electromagnetic Stirrer—- An electromagnetic stirrer (Iabline Inc.) was used in the formol,alcohol, and water media titration work. Glassware-- Volumetric glassware were of Pyrex glass brand. For large scale preparations the stainless steel equipment in the Michigan Department of Health Laboratories was used. Analytical Ultracentrifuggm- The Spinco Model E (Specialized In- st ruments Corp.) was utilized for studying the sedimentation behavior of proteins. 28. Materials and Reagents: Chemicals-- All inorganic and organic chemicals were either c.p. or reagent grade unless otherwise specified. Buffers- Phosphate buffers-- preportions were calculated using both the Henderson-Hasselbach equation and the Lewis ionic strength equation. Proportions for phosphate buffers used in Michigan Department of Health Laboratories were calculated by using industrial nomograms. In every case the pH was checked and adjusted with the aid of a pH-meter. SOdium acetate buffers-- Stock solutions of about 10 M acetic acid and about 1i M sodium acetate proved convenient in the preparation of the buffers . 1h Citrate buffers.- Stock solution of pH 6.0 ionic strength 0.3 buf- fer was prepared by adjusting 0.055 M sodium citrate solution to pH 6.0 with concentrated hydrochloric acid. Different pH—s were obtained by adjusting the stock solution with concentrated hydrochloric acid or Im so dium hydroxi de. Sodium glycinate buffer ij.5—- 75 gm. glycine and 20 gm.sodium hydroxide per liter. Glycine-acetate buffer pH 5.}... 150 ml.of 95 per cent ethanol, 2.0 ml,of M sodium acetate, 1.14 ml.of M acetic acid and 145 got“ glycine per litersg. Glycine—phosphate buffer pH 6.8 — 6.9-»- 160 ml. of 95 per cent ethanol, 1&5 g.of glycine, 2.5 m1.of sodium glycinate buffer) 3.2 ml.of O. 5 M di sodium phosphate and 2.“ ml.of 0.5 M monosodium phosphate brought to 1 liter?9' Buffers for electrophoresis: Glycine buffer pH 2.1-- ionic strengths 0.05 and 0.1. 0.05 M and 0.1 M hydrochloric acid respectively adjusted to pH 2.1 with glycine. Veronal buffer-- pH 8.6 ionic strength 0.1 was prepared by dissolv- ing 21.197 gm.of Veronal (Barbital, N.F., Fisher Scientific Company) and 0.1 mole of sodium hydroxide in distilled water and making the volume to 1 liter. Borate buffers-- proportions were calculated using the Henderson - H3338]. bach equation. 15 0.05 N Alcoholic Potassium hydroxide-- 3.75 gm.potassium hydroxide was dissolved in 62.5 ml. distilled water and diluted to one liter with 95 per cent ethanol. The reagent was standarized against 0.1067N hydro- chloric acid with phenolphthalein as indicator. Thymolphthalein Indicator-- The indicator solution for the Will- statter and Waldschmidt - Leitz(l92l)6o titration was prepared by di- luting 6 m1.of a 0.5 per cent thymolphthalein in 95 per cent ethanol to 100 ml with absolute alcohol. Saturated ammonium sulfate solution-- prepared by shaking at about 23° C until definite precipitate stayed on the bottom of the flask, then filtered. Since saturation depends on the temperature, N Molar solutions were used later. Proenzyme source-- Fractions III were received as frozen pastes from the American National Red Cross from stocks maintained at Squibb and Sons. The procedure for alcohol fractionation of pooled human plasma is summarized in Scheme 1. The purified proenzyme was stored frozen in 0.05 or 0.1 M acetate 'buffer pH “.6 or in water, acidified with a couple of drops of N hydro- chloric acid (pH around h.0). Casein Stock solution was prepared of Hammersten casein or Sheffield, 000m * m .“E “OHflgflom “O‘Hafifi IHflHmQOS Hog—Pm .80“me HHH .Hh HQHROH 3 H». lit PI . QHAM on» 6% N ofioflom on MideMMMuod Onaundooaorm economc eoeoo: o 33.8””: wages; 9:30.23 .28 AHHH coheoeuav use segue h 89C couch» a2 use 2) Modification of the Kline method“. a) Modified first step, buffer extractions compared with those of acid extracts.» citrate buffer of pH 8.6, I72 = 0.3 was used instead of 0.05 N sulfuric acid for M5 minute extraction of ly0philised 02-2 at room temperature and the data is given in Table XXII. The same lyo— philized paste was extracted at room temperature with 260 ml.of 0.05 M acetate buffer of pH M.6 for M5 minutes and centrifuged also at room temperature. Data showing effects of these buffers instead of 0.05 N sulfuric acid are given in Table XXII. When 500 ml.of 0.05 M acetate buffer pH I4.53 at 0° 0 was used for extraction of 02-2 (lot #17145) the results are given also in Table XXII. b) Partition of the proteins in 0.05 N sulfuric acid extract with saturated ammonium sulfate solution and subsequent activity is shown in Table XXIII. TABLE XXII Proteolytic Activity and Nitrogen in Buffer Extracts of Lyophilized Paste 02-2 Scheme 2 :— - Product P.U./m1. mg.N/m1. P.U./mg.N 8 Recovery 8 Total of Activity N 190 m1. citrate buffer pH 8.6 I72 = 0.3 230:5)“ 1.67 13.3 80 7.3 260 ml- acetate buffer pH M.6 u = 0.05 10.1 1.029 9.8 52.M Ext. 11250n 23.9 1.21 19.7 MM.7 801. 1 15.7 0.M35 36.1 26.3 Ppt. s 9.0 0.159 56.3 21.2 Super 3 3.8 0.1141 27.1 6.2 Both samples were from lot #1711 500 ml-acetate buffer a pH 11.53 M= 0.05 19.9(1:5) 0.M6 M3 80 Ext. 3280“ 13.3 0.371 36 M1 The sample in acetate buffer pH M.53 was left overnight. 300 m1 . of 0.05 N 11280)4 was used for extraction, pH 1.8. 5.3 The proteolytic activity was determined in 1 z 5 dilution. - n;- 51 TABLE XXIII Partition of Proteins with Saturated Ammonium Sulfate in Acid Extract of AM.2 Scheme M Starting Material pH RU/ml. M EU. (NHQ‘SOI, fig; 8 Recovery Time mlo mg.N satura—' mg.N of Activity ppt. tion Ext.3230h 1.9 1h.5 1.191 12.91 0-.20 1 hr. .20-.3M 60 19 overnight , 3) Modified second step in Kline Method. _ M" “'" Kim a) At pH 5.3 the precipitate after 3 hours standing was cen- trifuged, resuspended in 0.05 N sulfuric acid, and left at 0° 0 overnight. The next morning it was centrifuged in refrigerated centrifuge at 0° 0 to give the supernatant containing plasminogen called Solution A. The proteolytic activity and nitrogen loss in supernatant at pH 5.3 is given in Table XXIV. Specific activity of plasminogen in re-extracted 0.05 N sulfuric acid is presented in Table XXV. TABLE XXIV Proteolytic Activity and Nitrogen in Supernatant of Modified Kline Method at pH 5.3 ‘ nu/nu. mg.N/m1. 2g, 8 Total 8 of Total Ins]! Activity 1! _ 0.5 0.072 6.3 1.M 0.7 0.M 0.073 5.0 1.2 0.6 0.8 0.085 9.9 2.7 0-9 b) When solution A.of high specific activity (obtained by centrifuging the precipitate in second.step at pH 5.3 in Kline method and redissolving in 0.05 N sulfuric acid), was adjusted to pH 8.6 and precipitated with 0.02 M phosphate buffer pH 6.0, a new precipitate re- sulted with no increase of proteolytic activity as seen from Table XXVI. 52 TABLE XXV Proteolytic Activity and Nitrogen in Solution A of Modified Kline Method 207m. mg.N/m1. III/mg.N 8 Total 8 of Total Activity N 15.3 0.1M1 108.5 M3 1.5 21.5 0.197 109.M MM 1.3 i TABLE XXVI f Proteolytic Activity and Nitrogen in Precipitates B and Supernatants I B in Modified Kline Method 1 Product pH PU/ml. mg.N/m1. EU/mg.N' % Total % Total Time Activity N Ppt. Ppt.B 7.5 11.2 0.1M2 101 21 0.9 overnight Ppt-B 7.2 8.2 0.078 105.5 M0 1.2 11/2 hrs. Super B 7.5 1.5 0.038 MO.5 M.2 0.3 overnight SuperB 7.2 1.3 0.026 M8.5 1.7 0.1 11/2 hrs. Therefore ammonium sulfate fractionation was tried to increase the specific activity at the Solution.A stage. The specific activities of starting solutions A and ammonium sulfate fractions derived therefrom are given in Table XXVII. c) Experiments were made to further purify the precipitate B of plasminogen. Tables XXVIII and XXIX show the conditions of starting solutions subjected to various concentrations of ammonium sulfate to produce precipitate with resulting proteolytic activity of the products. Precipitation of plasminogen (or formation of ppt.B) in the last step of the Kline method was investigated in presence of 1 M and 0.1 M sodium chloride. In both cases the specific activity was decreased without increasing the yield. Centrifugation at pH 9.0 before precip- itation of plasminogen, as suggested by Kline, increased the activity at the expense of the yield. Specific Activities of Solutions A and of the Ammonium Sulfate TABLE xxv11 Fractions of Them 53 Solution A pH RU/ml. .11.. .221. (Nflh) 280M mg.N Saturation PM} 8 Recovery Time of mg.N Activity ppt. Scheme 2 2.0 (1ot #17M5) Scheme 2 Scheme 2 8.6 Scheme 2 2.0 Scheme 2 2.0 (Iot #1711) 7.0 A3-3 1.71 21.5 10.7 8.1 11.M b09 89 7.M 83 33-7 59 59 2.6 59.7 20 8 8.2 55 89 38 Recovery of activity is given as per cent of Solution A 1M-2‘ 2.0 AM-3(1) 1M.M 16.3 A6—3 1.85 Ab—3 16.3 2.2 7-5 6.8 10.3 12.M 0.197 109 0 -.20 M7 .20-.3M , 129 109 0 -e20 “8 0.098 .20—.3M 117 .1o-.3M 131 0 -.25 .25-.3M 1M5 .3M super 0 “e20 32eu .20.. 28 119.8 .28-. 3M 15M.0 0.22M 36e2 0 -e?5 .25.. 28 .28-.3M 95.6 Super .3M 0.132 86 0 -.20 21.8 0%.” 12 .8 0&031‘ 12 .7 .28-.3M 133.0 0.087 86 0 -.20 .2o-.3M 127.9 0.108 63 0 -.20 .20-.3M 115.3 .20-.3M 121.0 0 -.20 .2o-.3M 136 0 -.20 .20-.3M 128 0.11 95.5 0 -.20 - .20-.3M 106.7 Gees” “9.0 0 -020 e20”e3’+ lu3eh 36 3M 1+3 33 M5 min. overnight 1 hour 2 hours 1 hour overnight ; 1 hour :7 overnight f 1 hour 1 hour overnight overnight overnight 3 hours overnight overnight 2 hours overnight overnight overnight 1 1/2 hours overnight 2 hours M hours 1 hour overnight Vg’From AM-2 on, the recovery is given as per cent of Pr. III. M M (“HM)25°h was used throughout the experiment. 5M TABLE XXVIII Specific Activities of Precipitates B and Ammonium Sulfate Fractions of Them a; pH pH of EU. (NH ) 50‘ EU;'% Recovery Time Starting Material (NHu)QSOu_mg.N Saturgtion.mgtN Activity ppt. Ppt.B(lot #1711) 8.6 7.0 M9.3 Super .29 59.5 57 room to Ppt.B Super .25 55.M 28 left at 0° c Ppt.B 7.M5 7.0 Super .20 56.0 33 Ppt.B Super .25 39.M 13 Ppt.B 6.0 5.2 Super .20 63.1 M9 Ppt.B Super .25 57.9 27 Ppt.B 2.0 5.2 Super .20 59.8 60 Ppt.B 7.0 7.0 M5.6 Super .20 M7.3 51 overnight Ppt.B .20-.28 85.0 33 Ppt.B Sgper .28 71.M 12 Ppt.B 60.3 Super .20 53 M7 Ppt.B .20-.28 75.1 25 Ppt.B Super .28 20 Ppt.B 59e8 Super e20 b7e3 n6 Ppt.B .20-.28 81.M 2M Ppt.B Super .28 16.5 16 Ppt.B 51.2 Super .20 76.M 73 Ppt.B .20-.28 85.M 29 Ppt.B Super .28 38 Ppt.B 7.0 7.0 3M.3 0 -.10 83 overnight Ppt.B 0 -.15 83 Ppt.B 0 -.25 21 Ppt-B 0 -e28 701 Ppt.B 0 -03“ 1e5 Ppt.B Taken to 2.5 76.0 0 -.20 36.M 21 3 hours Ppt.B Taken to 7.2 .20-.28 82.M M7 overnight Ppt.B .28-.3M 30.6 2 Ppt.B Super .3M 2.6 Ppt.B 6 mo. in 2.0 73.7 0 -.20 6M 30 1 hour deep freeze 861 used .20-.28 10M 53 overnight .28-.3M 6M.7 15 overnight Ppt.B 6 mo. in 2.0 0 -.20 1 hour dBOPf'reeze .20-.3M 88 5 hours Ppt.B 6 mo. in 2.0 O -.20 68.2 31.1.3701 1 hour deep freeze HéSOu used .20-.28 11M.2 30.9901 1 1/2 hrs. .2 c.3M 101.2 RU.1569 ““— 55 TABLE XXIX Proteolytic Activity in Various Ammonium Sulfate Fractions Precipitated from.Acetate Buffer (NHh)280h EU. 8 Recovery Time EU. Saturation mgLN Activity ppt. Starting Material r72 29 waml. pH EL- 22.1 ... Acetate ‘ gelatinous buffer 0.05 M.53 92 0-.05 mass 2hrt. Ppt.B 1Yr .05-.2M 57.1 7.M overnight in deep freeze .2M—.3M 105 Acetate buffer 0.025 M.6 0-.25 123 9.2 0-.3M 122 22.5 .25..3M 1M8 1M.2 Super .3M 1.7 Acetate buffer 0.05 16.11 0.15 M.6 107 0-.20 32.7 5.2 11/2 hrs. 0 -.25 81.1 M3 1 1/2 hrs. .20-.25 78.1 25 .20-.28 93.2 50 .20—.3M 98.2 67 .25-.28 57.6 16 .25..3M 5M.3 M.7 .28-.3M 66.3 6.5 .28-.3M 66.6 12.5 .25-.28 M8 8.8 .25..3M 93.8 3M .28...3M 63.7 1M.5 Acetate buffer 0.05 17.16 0.256 M.55 67.0 0..15 36 13 3hrs. .15-.28 M2.6 M3 over weekend .28-.3“ 9 18 “ms. .3M..5o M6.2 2.6 Acetate pH 8.6 buffer 0.05 8.6 125 0-.20 78.8 M2 11/2 hrs. .20-.3M 1M 29 overnight 56 To concentrate the activity, 2.5 per cent TGA precipitation was tried but without success. Noteworthy is the observation that when dis- solved frozen precipitate B was thawed slowly at +0.50 0, with 0.1 M acetate buffer pH M.6 (equal amount added), and left overnight near 0° 0, the centrifuged supernatant increased in specific activity as shown in Table XXX. TABLE m T The Yield and Purity of Precipitate B Dissolved in Acetate Buffer E pH M.6 - “1" Starting Material fl Recovery Ell/mg.N of Activity P.U./m1. mg.N/m1. BIL/mg.N 26.3 0.3M5 76 106 27 30.9 0.309 100 107 29 56 68 a3Per cent recovery of starting material Fraction III. When a sample of frozen A3—3 (Scheme 3) was dissolved in 0.1 M acetate buffer pH M.6, 16.5 P.U./mg.N was increased to MM.6 P.U./mg. N. Sometimes P.U./mg.N were increased also by adjusting the slowly thawed dissolved precipitate B (78.8 P.U./mg. N) to pH 2.0: then the cen- trifuged supernatant showed 86.3 P.U./mg. N. In an attempt to recover specific activity of redissolved precipi- tate B in 0.05 M acetate buffer of pH M.6, experiments were carried out with the results shown in Table XXII. A solution of Precipitate B in 0.05 M acetate buffer of pH M.6, having an activity of 125 P.U./mg.N, gave a precipitate with hydrochloric acid-acetone (1 i 5) with 60.8 P.U./mg.N, and representing a M9 per cent recovery of activity. With 2.5 per cent TCA the specific activity ob- tained was 65.9 P.U./mg.N and indicated a 53 per cent recovery of activity. 57 '7‘ IN anaaenncez on» ere: acumen occuoon on anaraooe mo auo>ooom one snaoneo< camaooAm I." lull ”MNK HAMdH { a: ma mo.o :.m groom :1 mm no.0 :.m .eam m.- mmN.o m.m 00H :m moo.o :.~ .nnm o.Hm mac.o m.c :. om Hm moo.c :.m henna ~.m: m-.o w.m H.m Hm hm Mm.o hunch mm NN MN.O ...N .anmtq mm mm am.o noanm ma m am.o :.~ .acm mm a: ~:.c endow ca ea ~:.o :.~ .ucm ca H.o om mm moo.o :.~ .aam m: am mac.o :.~ .nam 4 a a hoe .2 Son m mm mm :H.o :.~ neasm A mm m :H.o :.~ .ncm mm mm m.o :.~ endow mm con ~.o :.~ Sam A mod mm~.c m.~a m: mm no.0 :.m noacm mma mm mm mc.o :.m .ecm mud on am no.0 :.~ .nca mma mma.c MN m.: z.ma\.p.m .2520... .23: me ahaoaeo4.uo _r.u.p. .» myopooem m. z.ws\.b.m .— mm 93695 Aegean: magma; 58 G. Plasminogen activity on TAMe and LE0 hydrolysis. Results of investigation to separate TAMe (p—toluene-sulfonyl-L~ arginine methyl ester) and L30 (lysine ethyl ester) activities by vari- ous treatments of plasminogen preparations are shown in Table XXXII. The activities were determined by titration in formaldehyde16'l7. Spontaneous and S.K. activated TAMe esterase activity of plasminogen is shown in Figure 5. H. The Fibrinolytic Activity of Plasminogen. The experiment was carried out as previously described52. When plasminogen of 136 P.U./mg.N (3.7 P.U./ml) was mixed with 0.M per cent protein (59 per cent clottable protein) no clot was formed. A control sample without enzyme but with the same amount of S.K. as in previous experiment, produced hydrolysis of the clot in 2 hours. In the second run of samples all the other conditions were kept the same as above but in.p1ace of 5 ml.of 0.M per cent protein, 1 m1. of higher clottability and higher fibrinogen content protein was used. With plasminogen of the above stated specific activity the clot lysed in 2 min. and M5 sec. tJhereas the control lysed in 2 hours. When a clot was formed in presence of cysteine, then there was very little hydrolysis even after 2M hours. Clot produced in the ab- sence of S.K. and enzyme showed very little hydrolysis during 3 days. .1. Physics-Chemical properties of the Highly Active Plasminogen. Ultracentrifuge sedimentation coefficients of the final plasminogen Preparation are given in Table XXXIII; the untracentrifuge patterns are seen in Figure 6. Electrophoresis patterns of plasminogen preparations with increasing proteolytic activity are presented in Figure 7. men and m.: we onerous on ceoaoooac oouaaaaooee mam emu mma oeeaooe : Hc.c :.~ ma he conceaAaooea mommaemzc em.-oa. new no man man sommasmzc :m4.m~i men an mam mma zomwhgmnc :m.to mom om me: man m.m we :cmmaamzc :m.-¢~. coo an Nam mm m.m_ma zommhamzc o~.to m.: we pounce oncaooc mmm mm ozm mma m.: we homage onoooo< on mmm m: .uad :.~ .. 0mm ma .eaa o.m on noses ma m.: we tenure honoree mm ca .scm m.m on come» mm m.: we acumen ooeaeo< mm." mm henna m.m 0» needs» ma mi ma young. succeed can moo mod cease m locum mo 0233.3 3 :8 353 :mfmod 9.: mm .333 003004 on own aoa : mc.o m.: we pounce onenooq coeaaonean anaoaoo4 oaqe couaaoecan r u.ws nod aeoonoecon. z.ms hoe noaneeecoem .3 as. one. no» on: a... 2035.." V. on; one 6:49 co sna>auoa unsound "Hume agm