University of Southern California Dissertations and Theses

Purification and identification of trypsin inhibitors in human serum

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Purification and identification of trypsin inhibitors in human serum

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Content PURIFICATION AND IDENTIFICATION OF TRYPSIN INHIBITORS IN HUMAN SERUM by Mai Young Park A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (Biochemistry) June 1964 U N I V E R S I T Y P A R K L O S A N G E L E S . C A L I F O R N I A 9 0 0 0 7 This dissertation, ivritten by MAI YOUNG PARK under the direction of h^T....Dissertation C o m mittee, and approved by all its members, has been presented to and accepted by the Graduate School, in partial fulfillment of requirements for the degree of D O C T O R O F P H I L O S O P H Y c 7 ]. / £ & • • •• *->............................................. \^z. Dean Date....... Jane, 1964 DISSERTATION COMMITTEE .. p Chairman C /> . / ’ ( cl t > ACKNOWLEDGMENTS I would like to express my appre ciation to the members of my dissertation committee, Dr. John ' . V . Mehl, Dr. Walter Marx, and Dr. Bernard J. Haverback, for their guidance and support. I am gratefu.1 to each member of the Soroptimist International Club, Huntington Park, for awarding fellowships in my early years of graduate study. I would like to give heartfelt thanks to my friends, Dr. Sandra B. Schiffman and Mrs. Jane West, for their encouragement, advice, and efforts in correcting my English in this dissertation. TABLE OF CONTENTS Page ACKNOWLEDGMENTS .................................. ii LIST OF ABBREVIATIONS U S E D .................... v LIST OF T A B L E S .................................. vi LIST OF ILLUSTRATIONS........................... vii Chapter I. INTRODUCTION ............................ 1 II. HISTORICAL REVIEW ..................... 3 III. MATERIALS AND METHODS................. 11 Materials Methods Trypsin Assay Procedure Total Trypsin Inhibitor Assay Procedure Trypsin-Binding Protein Assay Procedure The Stable Inhibitor Assay Procedure Treatment of Plasma Ion Exchange Chromatography Protein Determination Sephadex Column (Gel Filtration) Paper Electrophoresis Starch Electrophoresis Vertical Column Electrophoresis Ultracentrifugal Studies IV. EXPERIMENTAL RESULTS ................... 33 Determination of the Stable Inhibitor Acidification and Neutralization of Samples Effect of Heating on the Inhibitor Content of Samples Estimation of the Stable Inhibitor in Serum iii Chapter Chromatographic Separation of Three Serum Proteins pH Stability of the Stable Inhibitor Further Purification of the Stable Inhibitors DEAE-Cellulose II Column pH and Temperature Effects on the Stable Inhibitor "^Protein Fractionation with Zinc Ion TCA Treatment Sephadex G-100 Ultracentrifugal Studies Paper Electrophoresis Vertical Column Electrophoresis Gel Filtration Effect of TCA and Zinc Ion on the Stable Inhibitor Identification of the Stable Inhibitor Starch Electrophoresis Interactions of the Stable Inhibitors with Other Proteins Conversion of One Inhibitor to Another V. DISCUSSION ............................ VI. SUMMARY ............................... BIBLIOGRAPHY LIST OF ABBREVIATIONS USED The following abbreviations are used without definition: DEAE . . . diethylarainoethyl CM . . . . carboxymethyl TCA . . . trichloroacetic acid Tris . . . Tris (hydroxyinethyl) aminomethane rpm . . . revolutions per minute rps . . . revolutions per second psi . . . pound per square inch s 0 ,w OD mg Hg cm mp ml P1 ma sedimentation coefficient in water at 20° C Svedverg unit of sedimentation coefficient (10"13 sec) optical density milligram microgram centimeter millimicron milliliter microliter milliampere v LIST OF TABLES Table Page I. pH Changes in Trypsin and Trypsin Inhibitor Assays ..................... 34 II. Effect of Heating on the Stable Inhibitor Content of Serum ......... 36 III. pH Stability of the Inhibitor Fraction Obtained from DEAE-I Column ......... 41 IV. Effect of pH and Temperature on the Fraction from the DEAE-II Column . . 46 V. Fractionation of the Stable Inhibitor with 0.02 M Zinc Ion at Different pH Values.............................. 48 VI. Ultracentrifugation of Fractions from TCA Treated and Sephadex G-100 after TCA Treatment.......................... 53 VII. Ultracentrifugation Using Partition C e l l ................................... 54 VIII. Paper Electrophoresis of Inhibitors from Plasma, DEAE-I and II, and TCA Extract............................ 57 IX. Paper Electrophoresis of Fractions Obtained from Sephadex G-100, TCA, and DEAE-II............................ 58 X. Paper Electrophoresis of Fractions Obtained from Sephadex G-100 after DEAE-II Column ....................... 65 XI. Analysis of the Zn-TCA and TCA Treated Stable Inhibitor on Paper Electrophoresis ....................... 66 XII. Studies of Serum Protein-Inhibitor Interactions . 70 XIII. A Fraction from DEAE-II Column .... 71 XIV. Inhibitor Fraction from TCA Treated . . 72 XV. Studies on the Interconversion of One Inhibitor to Another ................ 73 vi LIST OF ILLUSTRATIONS Figure Page 1. The Stability of Inhibitor in Serum . . 37 2. Chromatographic Separation of Trypsin-Binding Protein, o(,-Inhibitor, and the Stable Inhibitor on DEAE-Cellulose Column ................ 40 3o Purification of the Stable Inhibitors . 43 4. Purification of the Stable Inhibitor on DEAE-Cellulose II Column ..... 44 5. Chromatography of a Partially Purified Stable Inhibitor in Sephadex G-100 Gel Filtration . .................. 51 6. Paper Electrophoretic Patterns .... 56 7. Vertical Column Electrophoresis of Serum (1.5 m l ) ....................... 59 8. Vertical Column Electrophoresis of DEAE-II Fraction ..................... 60 9. Vertical Column Electrophoresis of the Stable Inhibitor Fraction from Sephadex G-100 Gel Filtration after TCA Treatment......................... 62 10. Sephadex G-100 Gel Filtration ......... 63 llo Starch Block Electrophoresis of Serum . 58 vii CHAPTER I INTRODUCTION A number of naturally occurring trypsin inhibitors have been found in different tissues and in various species. Several of these substances were obtained as crystalline proteins and well characterized with respect to their physico-chemical properties. It is hoped that the investigation of the nature of trypsin inhibitors i_n vitro will eventually lead to the understanding of the physiological significance and the role of the trypsin inhibitors iri vivo. Human plasma is known to contain at least two trypsin inhibitors. Shulman (29) showed that there is one component which inhibits trypsin and another which inhibits both trypsin and plasmin. The first of these is quite readily inactivated by heating, and the latter is relatively stable. Jacobsson (12, 13) reported that two trypsin inhibitors from human serum were separated by electrophoresis on filter paper. One of these migrated as an ^-globulin while the second, a slower moving component, was identified with the ^-globulin fraction. Recently, Haverback and Dyce (11) reported the presence of a component in plasma which combines with trypsin in such a way that the 1 2 trypsin is still active and can no longer be inhibited by soybean inhibitor or the plasma inhibitors. Thus, there are at least three fractions in blood which react with trypsin. Two of these are inhibitors, which can be distinguished on the basis of stability to heat and acid. By far the largest amount of material is an d(|-globulin which represents the unstable inhibitor (o/j-inhibitor) . The stable inhibitor fraction is present in much smaller quantity. The third fraction is an ^-globulin, trypsin-binding protein. This dissertation deals with the separation of the three active proteins on a DEAE-cellulose column, and the further purification and characterization of the stable inhibitor. CHAPTER II HISTORICAL REVIEW The first trypsin inhibitor was discovered by Camus and Clay (4) in 1897 from the observation that serum inhibited the proteolytic activity of trypsin. Launoy (18) found trypsin inhibitor in human, rodent, chicken, and eel blood plasma. These discoveries led to isolation and concentration of trypsin inhibitors, whose physiological and chemical properties were then studied. Landsteiner (15) appears to have been the first to recover the inhibitory activity in serum from a precipitate of saturated ammonium sulfate solution. Schmitz (32, 33) isolated a trypsin inhibitor from 5 liters of bovine plasma. The plasma was diluted with 1.5 liters of 0.25 N H9S0., and a fraction was im* T obtained between 40 and 70 per cent saturation with ammonium sulfate. The dissolved precipitate was mixed with an equal volume of 5 per cent trichloroacetic acid and the clear filtrate was brought to pH 3.0 with 5 N NaOH. The inhibitor was precipitated by adding ammonium sulfate to 80 per cent saturation. A total of 20 to 30 mg of inhibitor was obtained. The inhibitor passed through an ultrafilter membrane and was believed to be a polypeptide. Grob (9) prepared 40 mg of "antiprotease" from 5 litersof pig's blood in 1949, which was shown to be active in inhibiting trypsin. The same author (10) later analyzed Cohn's (6) frac tions for the activities of trypsin inhibitors and found the inhibitor activity mostly in the fractions IV-1 and IV-4 and to a lesser extent in fractions V and I. According to the studies of Oncley, et a_l. (23) 89 per cent of the fraction IV-1 consist of c^-globulin (lipoproteins) of molecular weight 200,000 and 10 per cent of ^-globulin of molecular weight of 90,000, while 46 per cent of fraction IV-4 consist of d2-globulin of 300,000 and 38 per cent of ^-globulin of molecular weight 90,000. Duthie and Lorenz (7) found trypsin inhibitor in both albumin and globulin fractions in sheep serum. Twenty per cent of the total activity remained in the globulin fraction precipitated by 50 per cent satura tion with ammonium sulfate at pH 4.2, and 60 per cent in the albumin fraction obtained with saturated ammonium sulfate at the same hydrogen concentration. The antitrypsin factor was precipitated from serum with a loss of about 50 per cent by the addition of an equal volume of 50 per cent TCA, and no antitrypsin fraction was recovered in the filtrate. They also fractionated inhibitor from 1,5 liters of ox blood using Schmitz's 5 method. A total inhibitory activity of only 0.02 per cent of the original material was obtained in 180 mg. About 2/3 of the product was ammonium sulfate since the inhibitor could not be dialyzed. Martin (20, 21) isolated an enzyme inhibitor called "protease A" inhibitor from sheep serum by ammonium sulfate fractionation and chromatography on 1RC-50 resin. The inhibitor was homogeneous in electrophoresis at different pi I values and ultra centrifugation in phosphate buffer at pH 6.0. The isoelectric point was about 4.3. The sedimentation coefficient (s9q w) at pH 6.0 was reported to be 3.91 S. This inhibitor is an o((-globul in, unstable below pH 3.0, and inhibits trypsin, chymotrypsin, and plasmin stoichiometrically. Loomis (19) prepared an antifibrinolytic substance from bovine plasma. The plasma was fractionated between 50 and 80 per cent saturation with ammonium sulfate and the solution of precipitate was again brought to 50 per cent saturation with ammonium sulfate at pH 3.75. The precipitate, representing 60 per cent of original activity, was 85 per cent pure by electrophoretic analysis. Shulman (29) reported from "differential titra tion" studies that two trypsin inhibitors exist in human serum. He determined the plasmin inhibitor activity after saturating the trypsin inhibitor with trypsin, and the trypsin inhibitor activity was after inhibitor of plasmin was saturated with plasmin. The results showed one component which inhibits trypsin and another which inhibits both trypsin and plasmin. The first of these is quite readily inactivated by heating, and the latter is relatively stable. The trypsin and plasmin inhibiting substance was about 10 per cent of the total trypsin inhibitor. Shulman also attempted to separate the two types of inhibitors by means of ammonium sulfate precipitation. The best separation was found in the "two molar precipitate" which con tained 67 per cent of the plasmin inhibitor and only 11 per cent of the trypsin inhibitor that did not inhibit plasmin. The latter inhibitor was much less stable at 56° C than was the plasmin inhibitor. In the process of fractionation, 20 to 40 per cent of the original amount of inhibitor for each enzyme was lost. Subsequently, Jacobsson (12, 13) reported that two trypsin inhibitors from human serum could be separated by paper electrophoresis. One of these migrated as an dj-globulin while the second was identified with the ^-globulin fraction. He also presented evidence that the substance in the ^2- fraction is responsible for the inhibition of trypsin and of plasmin. The major inhibitor was in the ^-globulin fraction, which was about 90 per cent of the total trypsin inhibitors in serum. Shulman (30) purified trypsin inhibitor from human plasma and urine. Serum diluted with 19 volumes of distilled water was brought to pH 5.2 with 2 per cent acetic acid at 5° C. The precipitate was col lected, and the inhibitor was extracted with 5 per cent TCA at room temperature. The supernatant was saturated with ammonium sulfate. The dissolved precipitate in water was brought to 85 per cent ethanol concentration. The white flocculent precipi tate formed was dissolved in water, and then the solution was brought to pH 3.2 to precipitate the inhibitor. The inhibitor was heat resistant and TCA soluble, and also showed anticoagulant activity. The molecular weight of the urinary inhibitor was 16,400 from sedimentation and diffusion data and the inhibi tor from urine was homogeneous in ultracentrifugation. The inhibitor from plasma was inhomogeneous in ultra centrifugation and electrophoresis. The isoelectric point was 2.8 and that of the inhibitor-trypsin complex was 6.2. An amount of inhibitor equivalent to 0.748 mg of trypsin was obtained from 10 liters of plasma. 8 Moll, et. al. (22) obtained a thermolabile trypsin inhibitor from plasma of pregnant women in labor, by means of ammonium sulfate fractionation and anion exchange resin column chromatography. A twelve fold purification of trypsin inhibitor was obtained. The inhibitor was o(|-inhibitor and partially inactivated at pH below 5.0. At almost the same time, Bundy and Mehl (2) isolated a heat labile trypsin inhibitor with a method similar to that used by Moll. Starch block electrophoresis at pH 8.60 was used in addition to ammonium sulfate fractionation and anion exchange resin. The inhibitor was homogeneous in electrophoresis and ultracentrifugation. A hundred fold purification was obtainedo The sedimentation coefficient (son ) was 3.41 S at infinite dilution. £d\J , w The inhibitor was thermolabile and shown to be an ^i-globulin. Both trypsin and chymotrypsin were inhibited stoichiometrically by the inhibitor. Schultze, et (28) recently have identified this inhibitor with a plasma protein which they had isolated by another method and designated as the 3.5 S-o(|-globulin. Laskowski and his colleagues (16, 24, 34) have carried out purification of an acid labile trypsin inhibitor from bovine plasma during the last ten years. Recently Laskovvski and Wu (17) succeeded in isolating the acid labile trypsin inhibitor in crystalline form. The inhibitor fraction obtained between 50 and 60 per cent saturation with ammonium sulfate was chromato graphed on DEAE- and CM-cellulose ion exchange columns. The crystallization was carried out using a protein solution of 20 mg per ml with ammonium sulfate solu tion at 5° C. The sedimentation coefficient obtained was 4.0 S at pH 4.05 in acetate buffer, and 3.3 S at pH 6.85 in phosphate buffer solution. The absorbancy maximum was at 278 mp. The molecular weight obtained by diffusion and sedimentation method was 71,000 and by activity method was 39,000. Recently Haverback and Dyce (11) reported the presence of a component in the plasma fraction which combines with trypsin in such a way that the trypsin is still active and can no longer be inhibited by soybean inhibitor or the plasma inhibitors. When trypsin was added to serum and the mixture was sub jected to electrophoresis, a substance with trypsin like activity was detected migrating with c^-globulin fraction. We will refer to the protein which forms this compound with trypsin as trypsin-binding protein. In the usual inhibitor assay the trypsin-binding protein would appear to be an inhibitor. 10 In summarizing the literature, there are three active proteins in human plasma which react with trypsin. Two of these are inhibitors. One is the thermolabile, ^-inhibitor, while the other is stable at high temperatures and in acids. The third is a protein which binds trypsin, yet the combined trypsin retains a certain amount of tryptic activity. The results obtained with plasma of other species indi cated that trypsin inhibitors are widely distributed, but that the properties differ among different species. The occurrence of trypsin-binding protein has not been investigated except in humans. CHAPTER III MATERIALS AND METHODS Materials Trypsin.--Salt free, lyophilized, once crystallized trypsin was purchased from the Worthington Biochemical Company. The stock trypsin solution, 1 mg per ml of 0,0025 N HC1, was stored at 2° C. Working trypsin solutions were prepared by diluting the stock trypsin solution to the desired concentration with 0.0025 N HC1. Substrate.— A synthetic substrate for trypsin, benzoyl-arginine para-nitroanilide (BAPA) was pur chased from Mann Research Laboratory. A super saturated, aqueous substrate solution was prepared by heating a suspension of 1 mg of BAPA per ml at 80° C until dissolved, and then cooling it in water. The solution was filtered through paper to eliminate any undissolved BAPA which might initiate recrystal lization of the substrate. The substrate solution could be stored at room temperature for at least a month with no precipitation, and with little hydroly sis if stored in the dark. Occasionally the substrate solution became yellow due to hydrolysis, but it could 11 12 still be used for the routine analyses. Five per cent phosphotungstic acid.— The phosphotungstic acid was used as a protein-precipitating reagent and to stop the trypsin reaction. The reagent was prepared by dissolving 5 gm of phosphotungstic acid in 100 ml of 1 M Na-acetate buffer, pH 4.5. The acetate buffer was prepared by mixing 1 M acetic acid into 1 M Na-acetate solution until the pH of the solution reached 4.50. The phosphotungstic acid precipitates not only proteins, but the substrate. Tris buffer.— Tris (hydroxymethyl) aminomethane was purchased from Eastman Kodak Chemical Company. The buffer solution used for trypsin assay was 0.17 M Tris + 0.01 M CaCl9. The buffer was prepared by dissolving 26.6 gm of Tris in about 700 ml of distilled water, and then the pH of the solution was adjusted with conc. HC1 to 7.60-7.65. The volume was made up to 1 liter. Tris-citric acid buffer.— The buffer was pre pared by mixing 0.05 M Tris and 0.005 M citric acid until the pH of the solution reached 8.62. The buffer solution used in vertical electrophoresis was the Tris-citric acid buffer + 0.05 M NaCl. 13 Imidazole buffer.--The imidazol buffer was pre pared by dissolving 78 gm of imidazole in one liter of distilled water, and the pH of the buffer was adjusted with conc. HC1 to 6.0. For column chroma tography this buffer was diluted one to fifty with distilled water. Barbital buffer.— A solution of 0.1 i \ l sodium barbital was adjusted to pH 8.65 with conc. HC1. Plasma.--Human blood was obtained from a com mercial blood bank (California Transfusion Incorporated). Fresh blood was drawn with ACD solution. The blood was centrifuged at 1,900 rpm for thirty minutes after about one day of standing at 5° C. The supernatant was taken and used immediately or frozen. Saline.— Saline was a solution of 0.85 per cent NaCl. The pH of the solution was adjusted to 7 to 8 with 0.1 N NaOH. Rice starch sulfate.— Rice starch sulfate was prepared according to the method described by Bernfeld (1). EtOH-cellulose.— The ethanolized cellulose powder (Munktell's cellulose) was purchased from Grycksbo Pappersbruk AB, Grycksbo, Sweden. The powder 14 has good packing properties and very low adsorption capac ity. Sephadex G-100.— Sephadex G-100 gel was pur chased from Pharmacia Fine Chemicals, Incorporated, Rochester, Minnesota. Dialysis tubing.— The cellulose casing was purchased from Visking Company, Chicago, Illinois. The dialysis tubing was soaked about half an hour in water and washed once before use. DEAE-cellulose.--DEAK-cellulose was obtained from Eastman Kodak Company. The DEAE-cellulose was washed by the following procedure before and after use. A washing solution of 0.5 N NaOH and 0.5 M NaCi was added to DEAE-cellulose and the mixture was stirred vigorously and allowed to settle about four hours. After decanting the supernatant solution the DEAE- cellulose was washed with distilled water to bring the pH to neutrality. Then alcohol-acid solution (0.1 N HC1 in ethanol) was used to wash lipid out. The suspension was stirred and allowed to settle about four hours. The supernatant was removed, and washing by decantation with distilled water was continued until the pH of the washing solution was neutral. 15 Finally, the DEAE was rinsed with buffer to bring it to the desired pH. In order to shorten the time required for settling of the DEAE-cellulose, a sieve may be used. The mixture of DEAE-cellulose in alkaline solution was poured onto the sieve made with a screen and filter paper and washed with dis tilled water. Therefore, the fresh washing water was not mixed with the previous solution. Methods Trypsin Assay Procedure The activity of trypsin was measured by the hydrolysis of the synthetic substrate BAPA. The working trypsin solution, 0.2 ml, and 1.8 ml of the Tris buffer solution were added to a test tube. The mixture was preincubated for ten minutes in a 37° C heating block, then 1.0 ml of the substrate solution was added. After twenty minutes of incubation the digestion was stopped by adding 1.0 ml of 5 per cent phosphotungstic acid solution. The cloudy mixture was clarified by centrifugation and filtration through Whatman Number 1 filter paper. If the solution was still turbid due to unprecipitated proteins, the mixture was left at room temperature for thirty minutes and again centrifuged. The product, para- nitoaniline (PNA), formed during the hydrolysis was measured at 383 mp with a Beckman B spectrophotometer. Total Trypsin Inhibitor Assay Procedure The unit of inhibitor was defined as the amount of trypsin, in pg, combined with inhibitor. The amount of the combined trypsin was determined by comparing the activity of two trypsin samples: one containing trypsin as a standard, the other trypsin and inhibitor. For example, a trypsin standard of 14 pg gave an optical density (OD) reading of 1.000 at 383 mp and with the inhibitor in 0.2 ml of sample, an optical density of 0.500. Consequently, 50 per cent of the trypsin, or 7 pg, was inhibited. Since this represented the amount in 0.2 ml, the inhibitor in the original sample was equal to 35 pg of combined trypsin (CT) per ml. The value of trypsin inhibitor was not corrected for the amount of active trypsin present in the working solution. The assay procedure was similar to that for the determination of trypsin activity, except that inhibitor was added in place of part of the buffer. Inhibitor was added to a test tube, and the volume was made up to 1.8 ml with the Tris buffer described above. Then 0.2 ml of the working trypsin solution 17 solution was added. After hydrolysis was stopped by adding 5 per cent phosphotungstic acid, the residual trypsin activity was determined by measuring PNA formed. Trypsin-Binding Protein Assay Procedure The trypsin-binding protein activity was deter mined by measuring the tryptic activity in the presence of the binding protein, trypsin, and excess soybean trypsin inhibitor. An excess amount of soybean trypsin inhibitor means an amount of the inhibitor more than sufficient to inhibit the activity of the trypsin completely in the absence of the binding protein. The activity of the trypsin-binding protein is expressed as ug of trypsin giving an activity equal to that found in such a mixture. It is evident that the total amount of trypsin present must be in excess of the trypsin-binding protein. The general procedure for the estimation of the trypsin-binding protein was similar to the trypsin assay or the trypsin inhibitor assay. Aliquots of the trypsin-binding protein were added to test tubes and the Tris buffer was added to bring the volume to 2.0 ml after addition of the working trypsin solution. The mixture was left at 37° C for fifteen minutes to allow the trypsin to bind with the protein in the sample. Then the excess 18 amount of soybean inhibitor (20 yig) was added to each test tube and the trypsin activity was measured according to the assay procedure described. The blanks were prepared as above except the 5 per cent phosphotungstic acid was added to the test tube before the substrate solution. The optical density at 383 mp was compared with that of the standard. The Stable Inhibitor Assay Procedure The unit of the stable inhibitor was the same as the total inhibitor determined by the method described above. Normal serum was diluted with saline. Each test tube, containing 0.1-0.6 ml of diluted serum and 0.1 ml of 0.5 \! acetic acid buffer (pH 4.10), was placed in a heating block which was previously adjusted to 60° C. At specified time intervals, each tube was taken out of the block and was immediately immersed in an ice water bath for five to ten minutes. The volume of the solution was made up to 1.8 ml with the Tris buffer, pH 7.65. After adding 0.2 ml of working trypsin (14 pg) the reaction mixture was preincubated for ten minutes at 37° C in a heating block before lo0 ml of substrate (BAPA) was added. After twenty minutes of incubation, 1.0 ml of 5 per cent phosphotungstic acid was added 19 to stop the reaction. The precipitate was removed by centrifugation. The PNA in the filtrate was determined by reading the optical density at 383 mp with the Beckman B spectrophotometer. The assay procedure for studying the thermo-stability of the trypsin inhibitor was similar to the above except for the adjustment of pH during heating. Treatment of Plasma One liter of plasma was mixed slowly with rice starch sulfate dissolved in concentration of 2 gm in 100 ml of saline. The mixture was allowed to stand at 5° C for four hours to overnight with constant stirring. The supernatant was separated from the precipitate by centrifugation for thirty minutes at 1,900 rpm at 5° C. The precipitate was discarded. The supernatant was then dialyzed against 30 liters of the starting buffer for twenty-four to forty-eight hours at 5° C with constant stirring. One week to ten days were required from the time the blood was drawn until the plasma was ready for chromatography. Ion Exchange Chromatography The DEAE-cellulose was chosen for the initial separation of proteins, o^-inhibitor, trypsin-binding protein, and the stable inhibitor and for the further 20 purification of the stable inhibitor. Preparation of column.— The column was prepared by pouring a suspension of the DEAE-cellulose in the starting buffer into the column and packing under an air pressure of less than 5 psi. Development of chromatogram.--Two columns were used, one for the separation of the initial fractions and another for subsequent purification of the stable inhibitor. DEAE-cellulose column I (DEAE-I)— the large scale fractionation system was operated automatically: applying the sample to the column, changing different buffers to develop the chromatogram, recording the concentration of proteins in the effluent, and collect ing fractions in bottles. The treated plasma (1 liter) was applied on the DEAE-I which was previously equilibrated with the starting buffer until the pH and conductivity of the inflow and outflow were the same. The size of the column was 8.5 x 120 cm, and the column volume was 3.5 to 4.0 liters. The flow rate, including that during application of the sample, was 6.5 to 7.0 ml per minute. Fractions were collected for one-half or one hour periods. The buffer solutions used in this column were as follows: 1. Starting buffer— 0.06 M NaCl + 0.02 M. Tris buffer, pH 7.7 2. Second buffer — 0.12 M " + 3. Third buffer — 0.35 M " + " After all the sample was in the column, 10 liters of the starting buffer were used to wash out the unadsorbed proteins. Then a linear gradient was started using 3.5 liters of the starting buffer in the mixer and 3.5 liters of the second buffer in the reservoir. Both mixer and reservoir were open at the top, and were bottles of the same diameter, connected at the bottom. At the first salt gradient region, trypsin-binding protein was eluted and was closely followed by Oij-inhibitor. Actually the trypsin- binding protein was overlapped by o^-inhibitor. The elution of ofi -inhibitor was completed by applying the second buffer until the effluent showed an OD of 0.20-0.25 at 280 mp. About 10 liters of the third buffer were used to elute the stable inhibitor. DEAE-cellulose column II (DEAE-II)--the lyophilized stable inhibitor fraction from DEAE-I was chromatogramed again on the DEAE-cellulose column II with imidazole buffer, pH 6.0. The size of the column was 6.5 x 70 cm, and the column volume was 22 1.7-2.0 liters. The flow rate was 1.5-2.0 ml per minute. About 15-20 ml fractions were collected in an automatic fraction collector. For the elution of the column a salt gradient was applied. A reservoir contained stronger buffer and a mixer contained weaker buffer solution. Thus the con centration of salts in the mixer progressively increased as the elution proceeded. The salt con centration of the reservoir and mixer were calculated as follows: The inflow and the outflow of the mixer were made equal by sealing the mixer, so the column of the mixer remained constant. When a volume, dv passes in and out of the mixer, Xdv moles are added to the mixer from the reservoir, while xdv moles leave the mixer and enter the column. Then the net increase in moles in the mixer: dm = dv (X-x) Then the increase in concentration in the mixer: dx = dm = dv (x_x) --------------------- V V Where X = Cone, of reservoir x = Cone. of mixer v = Volume of mixer 23 If x is the initial concentration in the mixer, and o * x^ is the concentration at time t, when a volume V has entered the mixer from the reservoir, then the integral of equation (l) is v / 1 -V/v-v - V / v 0 x = \ (1-e ) + x e 2 t o Applying the equation, one can obtain a desired change of salt concentration by varying the volume of the mixer and concentration in the reservoir and the mixer in relation to the volume over which the gradient is to be developed. The following buffer solutions were used for the development of chromatogram: 1. Starting buffer— 0.15 M NaCl + 0.02 M imidazole, pH 6.0 2. Second buffer — 0.25 \ \ " + " 3. Third buffer — 0.59 M " + The protein fraction from DEAE-I was dissolved in 100 ml of the starting buffer, and the undissolved protein was centrifuged out. After applying the sample to the column, the column was washed with about 1 liter of the starting buffer to wash out the unadsorbed proteins. This was followed by a salt gradient from 0.15 M to 0.20 M NaCl by the method described above. Two liters of the second buffer were placed in the reservoir which was connected to the top of the mixer, containing 24 3 liters of the starting buffer, so that the mixer was sealed and the volume remained constant. To elute the stable inhibitor, 3 liters of the third buffer were in the reservoir, 2 liters of 0.20 M were in the mixer. Thus the gradient was continuously increasing as the stable inhibitor eluted. Protein Determination Protein was estimated by reading optical density of a solution at 280 mp with the Beckman DU spectro photometer . Sephadex Column (Gel FiTtration) Sephadex G-100 was suspended in a buffer solu tion (0.02 M Tris + 0.1 M NaCl, pH = 7.7), and the suspension was stirred for two hours with a magnetic stirrer. The supernatant was decanted after settling of the gel. The thick suspension was poured slowly into a column filled with the buffer, and allowed to sediment gradually. A filter paper was placed on top of the gel, and the sample was applied. The flow rate of the small column (1.0 x 25 cm) was 12 ml per hour at the beginning of the run and gradually changed to 6 ml per hour. Two ml fractions were collected. The flow rate of the larger column (2.8 x 100 cm) was 25 12 ml per hour throughout the run, and 3 ml fractions were collected. The protein content and the trypsin inhibitor activity were estimated in the fractions. Paper Electrophoresis The protein components of the plasma and certain plasma fractions were characterized by paper electrophoresis using a Spinco-Durrum apparatus. The Whatman thick strip paper was dipped into the buffer solution (Ool M sodium barbital, pH 8.65) and then pressed between paper towels to absorb the excess buffer before being placed in the apparatus. After- electrophoresis for seventeen to twenty hours, about 110 volts and 7-9 ma at 5° C, the strips used for the localization of proteins were dried at 100° C for twenty minutes and then stained with bromophenol blue for thirty to sixty minutes. Excess dye was removed by washing with 5 per cent acetic acid solution until the background became white. When it was desired to elute the proteins from the paper after electrophoresis, the paper strips were not dried but were kept at 5° C until they were cut into several segments according to the protein peaks. Each segment was cut into smaller pieces to make it easier to drop them into the test tube. One ml of Tris buffer for the trypsin assay was added to each test tube and the tubes were left at 5° C overnight. The eluate was assayed for trypsin inhibitor activity. Starch Electrophoresis Potato starch powder was purchased from J. T. Baker Company. The procedure of Kunkel and Slater (14) was followed. The starch powder, 500 gm, was washed four times each with water and the barbital buffer, pH 8.65, by mixing the starch with the washing solutions and decanting the supernatant. The washed starch paste was poured into a lucite block 5 x 37 and 4.0 cm in depth. The excess buffer on the surface of the starch paste was adsorbed by paper towels. The block was wrapped with parafilm and was placed at 5° C for at least four hours. Sample was applied in form of a paste obtained by mixing 3 ml plasma, equilibrated with the buffer by dialysis, with starch powder. The plasma was mixed with a small amount of bromophenol blue to observe the protein front, since albumin is known to combine with the dye in its native state. Electrophoresis was carried out for thirty-two to thirty-nine hours at 5° C with a voltage of 180 and a current of 14-16 ma. The protein pattern was 27 obtained by inserting a Whatman filter paper strip on its edge into the starch, and then staining the protein adsorbed onto the paper was as in the pro cedure used for paper electrophoresis. The starch was cut into 1 cm segments and each segment was put into a test tube. Five ml of imidazole-saline buffer (0.1 M imidazole + 0.85 per cent NaCl, pH 7.2) were added and the starch was well stirred and left at 5° C overnight. The supernatant was decanted and filtered. The remaining segments, number 1-5 counting from the cathode end, were eluted in 10 ml of the buffer. The protein content in the samples was estimated from the optical density at 280 m|i. The eluates number 1-5 were concentrated by lyophilization. The lyophilized protein dissolved in buffer and the other eluates were assayed for trypsin inhibitors and trypsin-binding activities. Vertical Column Electrophoresis Preparatory vertical electrophoresis was applied for the purification of the stable inhibitor with selected supporting medium and conditions. The appara tus was constructed according to a modification of Porath's (8, 27) method. It has the advantage of low cost, ease of operation, and flexibility. As a 28 stabilizing medium, Munktell's cellulose powder was chosen. Among other supporting media tried were Sephadex G-25, starch, and cellulose. Munktell's cellulose powder has good packing properties and a very low adsorption capacity. A Tris-citric acid buffer system was preferred as a electrolyte to veronal buffer system. Construction of the apparatus.— Fyrex glass tubes (2.8 x 40 cm) and ground glass joints 24/40 were used for the column and a bridge. The apparatus consists of three parts: a column, a connecting bridge from the column to the electrode vessel, and an adaptor from the column to a fraction collector. The column was joined to the U-tube of the bridge with the ground glass joint. The column and the other end of the bridge were immersed in polyethylene cylinders which are the same as electrode vessels. The adaptor was attached to the bottom of the column with the ground glass joint after electrophoresis to elute proteins. Packing in the column was supported with glass wool and a filter paper on top of glass rods across the column. Electrodes.— Electrodes were prepared using a platinum wire (75 cm) around a rectangular plastic 29 rod (40 cm). Polyethylene cylinders of 2 liters capacity were conveniently used for electrode vessels, since a long container to insert the column was necessary. The electrolyte used in this system was prepared by mixing 2 liters of 0.05 M Tris with 1 liter of 0.005 M of citric acid, and 0.05 M NaCl was addedc One buffer system was applied. Preparation and operation of the column. The powder was washed several times with the buffer described. A suspension of the powder was poured into the column and allowed to sediment gradu ally by gravity. The dimensions of the bed were 40 x 2.0 cm. After packing the column it was washed with buffer and then left at 0° C overnight for temperature equilibrium. Serum (1.5-2o0 ml) equili brated with the buffer by dialysis or lyophilized protein dissolved in the buffer solution was applied to the top of the column. After all the sample had entered the column, several ml of the buffer were applied until the sample reached a desired position. Serum dyed with bromophenol blue was used as a marker. The flow' of liquid through the column could be stopped by closing the bottom of the adaptor by a screw-clainp. 30 Electrophoresis.— The bridge was connected, and the adaptor was taken off the column. The column was quickly immersed into one of the electrode vessels filled with the buffer. Air in the bottom of the column and in the bridge was removed with a rubber tube. The levels of buffer in both electrode vessels were well balanced in order to prevent flow through the column. The voltage applied was 300-350 volts to maintain 30 ma for twenty to twenty-six hours. The pH of the buffer in both electrode vessels was expected to change during the electrophoresis, and was checked after the electrophoresis. It was found that the electrolytes could be reutilized, since mixing them together gave the original pH. Elution.— As soon as the current was cut off the column was taken out of the vessels, and the adaptor was attached. The buffer in the top of the column was discarded and fresh solution was added for the elution. The flow rate was 2.8 ml per ten minutes and 2-3 ml fractions were collected. The proteins and the inhibitor activities in the effluent were measured. The column was reused three times without repacking or regenerating the cellulose except for washing the column with the buffer. Ultracentrifugal Studies A Spinco Analytical Ultracentrifuge, Model E, was used in the sedimentation studies. Standard and partition cells were used at the speed of 56,100 rpm at 20° C for about three hours. Sedimentation coeffi cients were calculated from the schlieren patterns photographed four or five times per run, using the equation (31) In (X /X ) M + 0 x v | s20,w = 1 7 ~ t X 120 x 1w Where X? = Distance from center of rotation to peak in cm X. = Distance from center of rotation to meniscus in cm Id = Angular velocity in radians 2 rTrps t = Time in seconds (1/3 of acceleration + running time) 1t° x Is D o X r cone. ^ 1.000 at 20° C I20 x 1w Sedimentation coefficients were also calculated from the protein concentration and activity of trypsin inhibitor taken from the top of the partition at the end of the run by the method of Petermann, et. al. (26) cone. 32 In (C/CQ) x <Xo/Xl>] s 2 -W2 t t = Time in seconds (1/3 of acceleration deceleration time + running time) X = Distance from the center of rotation ° meniscus in cm X, = Distance from the center of rotation partition in cm and to the to the CHAPTER IV EXPERIMENTAL RESULTS Determination of the Stable Inhibitor Earlier work had shown that the o((-inhibitor is quite unstable to heating and to even a moderately low pH. Although it was clear that the amount of the stable inhibitor could be measured after inactivation of tfj-inhibitor, the exact time required for complete inactivation was not known, nor was the extent to which heating could be continued without destroying any of the stable inhibitor. Acidification and Neutralization of Samples A simple process of acidification of samples during inactivation and subsequent neutralization for the inhibitor assay was sought, particularly since it was hoped this would provide the basis for a routine analysis. A series of human serum samples, diluted ten times with saline, was mixed with 0.1 ml of 0.5 M Na-acetate buffer, which would bring the pH of samples down to about 4.0 and would be neutralized easily by addition of the assay buffer. Table I shows the pH values determined after mixing the diluted serum with the acid buffer and the pH which 33 34 TABLE I pH CHANGES IN TRYPSIN AND TRYPSIN INHIBITOR ASSAYS Diluted* Serum ml Acid* * Buffer ml Initial pH Assay * * * Buffer ml Final pH o o 0.1 4o08 1.6 7.10 0.2 0.1 4.08 1.5 7.10 0.3 0.1 4.08 1.4 7.10 0.4 0.1 4.08 1.3 7.08 0.5 0.1 4.07 1.2 7.05 *Serum diluted to ten times with saline. **0.5 M Na-acetate buffer, pH 4.10. ***0.17 M Tris buffer + 0.01 M CaCl2, pH 7.40. 35 was subsequently reached when the assay buffer was added to the acidified serum. Effect of Heating on the Inhibitor Content of Samples Vvhen the samples were heated at 60° C at a low pH, a certain amount of precipitate was formed. Since it was not clear whether a sample of clarified super natant could be taken for the assay, the amount of inhibitor recovered in the supernatant after centri fuging was compared with that in the mixture before the centrifugation. The turbidity in each tube was also recorded. The results shown in Table II indi cated that the amount of the inhibitor in the super natant was less than that in the centrifuged mixture. Since it appeared that some inhibitor was lost by occlusion in the precipitate, the total mixture after heating was used. Estimation of the Stable Inhibitor in Serum The method of measuring the stable inhibitor in serum has been described in Chapter III. The effects of heating for varying periods of time are shown in Figure 1, and compared with the effects of heating diluted serum in the absence of the acid buffer. The results indicated that the level of TABLE II EFFECT OF HEATING ON THE STABLE INHIBITOR CONTENT OF SERUM Heating Time in Minutes Turbidity pg CT/ml Serum* Supernatant Mixture Experiment Number 1 2 1 2 10 + 97 119 103 125 20 + 113 83 103 100 30 + + 97 68 110 100 40 ++ + 33 73 120 90 50 ++ + 20 53 97 - 60 ++++ 13 53 87 105 *The unit of inhibitor was amount of combined trypsin in jig expressed as the (pg CT). 37 ft- -ft 60 Vj i fj i) Figure 1.— The stability of inhibitor in serum. Q— -0 The amount of inhibitor left after heating diluted serum at 60 C with acid. •~-#The amount of inhibitor left after heating at 60 C only. 38 inhibitor reached after ten minutes at pH 4.1 was close to that reached after forty minutes at the higher pH. Furthermore, there was little decrease in activity beyond ten minutes, and between thirty and 120 minutes there was essentially no change. Chromatographic Separation of Three Serum Proteins Three active proteins in plasma were separated on a DEaE-cellulose column, which was operated as described in Chapter III. The optical density at 280 mp was determined for each bottle as a measure of protein concentration, and the activities of the trypsin-binding protein, c<( -inhibitor, and the stable inhibitor were determined in every other bottle. The fractions containing the high activity in each case were pooled. For the trypsin-binding protein, bottles number 27-35 were pooled to yield 3,300 ml. The pooled fractions contained the binding protein equivalent to 44,550 pg trypsin and proteins equivalent to 3,234 optical density units at 280 mp. The ratio of the activity to optical density at 280 mp (specific activity) was 15. The ^-inhibitor was pooled from bottles number 36-51 in 56,000 ml, dialyzed, and lyophilized for storage. 39 The stable inhibitor was pooled from bottles number 82-85 in 1,760 ml. The fraction contained 4,417 optical density units and inhibitors equivalent to 38,720 yg of trypsin. The specific activity of the fraction was 8.8. The recovery of the stable inhibitor in this experiment was 41 per cent of that estimated in the initial plasma sample. Distribution of the binding protein, inhibitors, and proteins is shown in Figure 2. pH Stability of the Stable Inhibitor The partially purified stable inhibitor from DEAE-I was tested for its stability as a function of pH. Three ml of the sample were used. The pH was adjusted with either 0„1 N HC1 or 0.1 N i\aOH, and the samples were then allowed to stand at 5° C for seventy-two hours. Then the pH was adjusted to neutrality. The volume was brought up to 5.0 ml with water, and each was assayed for total inhibitor. The results are shown in Table III. Sample number 6 was taken as the standard for calculation of per cent inhibitor left. This preparation contained about 40 per cent of the unstable, (^-inhibitor, which is known to become rapidly inactivated below pH 6, but which is relatively stable at higher pH 40 Figure 2.— Chromatographic separation of trypsin-binding protein, o(r inhibitor, and the stable inhibitor on DEAE-cellulose column. The starting buffer was 0.02 M Tris-HCl + 0.06 M NaCl, pH 7.7. 41 TABLE III pH STABILITY OF THE INHIBITOR FRACTION OBTAINED FROM DEAE-I COLUMN Tube Number pH Turbidity pg CT/3 ml of Sample Per Cent Inhibitor* Recovered 1 1.26 + + 85 65 o 2.43 + + 80 61 3 3.46 + + 87 67 4 4.45 + + + + - - 5 5.10 + 101 78 6 6.38 0 130 100 7 7.9 0 125 96 8 9.0 0 120 92 9 10.17 0 133 100 *Sample number 6 was used as a standard. Each sample was exposed to different pH values for seventy- two hours at 5° C. The amount of the inhibitor left was determined by the method described in the text. 42 values. The results indicated that there need be little restriction on the pH which might be employed in the further purification of the stable inhibitor. Further Purification of the Stable Inhibitors A general outline for purification of the stable inhibitors is given in Figure 3. DEAE-Cellulose II Column The lyophilized stable inhibitor fraction from DEAE-I was chromatographed again on DEAE-II in 0.02 M imidazole buffer, pH 6.0 with a salt gradient. The protein content and activities of the stable inhibitor and total inhibitor were determined in every other tube. Results shown in Figure 4 indicated that the major portion of the protein and a sub stantial amount, of inhibitor passed through the column with the starting buffer, but that this fraction contained no stable inhibitor. A second inhibitor peak appeared much later on the chromatogram. The total trypsin inhibitor activity in this case was equal to the activity of the stable inhibitor, and no trypsin-binding protein activity was recovered in this fraction. This step clearly completed the isolation of the stable inhibitor from c^-inhibitor 43 Plasma Defibrination Ppt. Sup. DEAE-I, pH = 7.7 Trypsin-Binding °(|-inhibitor Stable inhibitor protein DEAE-II Imidazole buffer pH = 6.0 1 + + Zn Fractionation Fract.I Fract.II i Sup. Ppt. TCA treatment Sup. Ppt. Sephadex G-100 6 Stable inhibitor Stable 'inhibitor fast-moving Vertical column electrophoresis pH = 8.65 Stable inhibitor slower-moving Figure 3.--Purification of the stable inhibitors. 44 Figure 4.— Purification of the stable inhibitor on DEAE-cellulose II column. The starting buffer was 0,02 M imidazole + 0.15 M NaCl, pH 6.0. A salt gradient 0.15 M to 0.5 M was applied to elute the protein. 45 and trypsin-binding protein. The fractions with high activity were pooled, dialyzed, and lyophilized. On the average, 29,000 yg CT were recovered from 1 liter of serum. The recovery was about 55 per cent of the fraction applied on DEAE-I I < , The specific activity varied in the range of 40-90. pH and Temperature Effects on the Stable Inhibitor This experiment was designed to determine whether the stable inhibitor fraction from DEAE-II was devoid of -inhibitor and to study the stability of the stable inhibitor at different pH values and temperatures. The buffer used in this experiment was a mixture of three different buffers in order to eliminate the effect of buffers on the trypsin inhibitor assay. The buffer was made to 0.05 M in Tris, imidazole, and Na-acetate, and the pH was adjusted to 7.99, 6.04, or 3.95 with conc. HC1. Aliquots of the sample were diluted with each buffer to specific pH values and then were placed in the 60° C and the 37° C heating blocks for certain time intervals. The assay pro cedure used was described in Chapter III. The results are shown in Table IV. 46 TABLE IV EFFECT OF pH AND TEMPERATURE ON THE FRACTION FROM THE DEAE-II COLUMN Time in Minutes Per Cent Inhibitor Recovered pH 4 pH 6 pH 8 37° C 60° C 37° C 05 o o o 37° C 60° C 0 100 100 100 100 100 100 10 98 93 104 96 102 100 20 95 96 104 104 99 98 30 98 93 104 93 102 100 40 100 93 100 98 98 102 50 96 93 100 93 104 94 60 94 89 104 96 102 96 Note: The fraction from the DEAE-II column was examined for its pH stability. The fractions were exposed to pH'sQ4, 6, gnd 8 for a series of time intervals at 37 or 60 C. The inhibitor was stable under the conditions used. 47 Protein Fractionation with Zinc Ion Fractional precipitation of protein with zinc ion was studied as a possible step in the purifica tion. The effect of hydrogen ion on the precipitation of the inhibitor by zinc ion was investigated. Twenty-five mg of the stable inhibitor fraction from DEAE-II were dissolved in 25 ml of saline. Four ml of the solution were mixed with 1 ml of 0.1 M Zn-acetate solution to give a final zinc ion con centration of 0.02 M. (The saline was stored at 0 i4 5 C, and the Zn-acetate was at room temperature.) The pH of the mixture was adjusted with 1 N HC1 or 1 N NaOII on a Beckman pH meter. The mixture was allowed to stand at 5° C for four hours and was centrifuged at 3,000 rpm for twenty minutes at room temperature. The precipitate was dissolved in saline with the aid of a few drops of 2 per cent versene solution and the pH was adjusted to 7-8 with 1 N NaOH. The activity of the stable inhibitor and the protein content of the supernatants and the precipitates were determined. The results, shown in Table V, indicated that the stable inhibitor was completely precipitated at about pH 6.62 with 0.02 M Zn^ ion. There were considerable amounts of the stable inhibitor recovered TABLE V FRACTIONATION OF THE STABLE INHIBITOR WITH 0 o 02 M ZINC ION AT DIFFERENT pH VALUES pH OD 280 mp pg CT/ml pg CT/OD 280 mp Volume ml Total Inhibitor Recovered orig. .978 54 55 8 432 Sup. 5.25 .418 35 83 9.4 329 5.62 .442 34 79 9.7 340 6.22 .282 26 93 90 4 245 6.62 .022 0 0 — — 7 oOO .022 0 0 — — 7.62 .022 0 0 — — 8.18 .022 0 0 — - Ppt. 5.25 — 21 — 7.7 162 5.62 — 17 — 80 3 141 6.22 " 27 " 9.3 251 Note: Protein fractionation with zinc ion was carried out at different pH values. The amounts of inhibitor left in the supernatant and the precipitate were determined. A pH range of 5.25 to 5.62 would be used for the purification of the stable inhibitor. 00 49 in the precipitates at pH values below 6.22. However, the specific activity of the inhibitor in the super natant was increased by about two fold in this experiment, and the average specific activity obtained from this step was 100. TCA Treatment The stable inhibitor fraction from DEAE-II with specific activity 78 was dissolved in saline and the denatured protein was removed by centrifuging for twenty minutes at 3,000 rpm. After adding 0.1 M Zn-acetate to a concentration of 0,02 M Zn, the pH was adjusted to 5o62 with 1 N HC1. The mixture was left at 5° C for four hours. Then the precipitate was centrifuged, and the supernatant was treated with 10 per cent TCA to give a final concentration of 2.5 per cent TCA. After about three hours at 5° C the supernatant was removed by centrifuging the mixture for thirty minutes at 10,000 rpm. The specific activity of the supernatant was 307, and 2,726 pg CT were obtained from 1 liter of plasma. With one more treatment with 2.5 per cent TCA the specific activity was increased to 427, but the yield was low: 1,845 pg CT equivalent was obtained. Sephadex G-100 Sephadex G-lOO was utilized to further purify the stable inhibitor which had been treated with ZrT ion and TCA. The lyophilized protein having inhibitor equivalent to 1,845 pg with a specific activity of 427 was dissolved in 1 ml of buffer (0.02 M Tris + 0.1 M NaCl) with which the column had been prepared. The column was developed using this buffer, and 2 ml fractions were collected at a flow rate of 0.1 ml per minute. Protein content and the " inhibitor activity were determined. The results are shown in Figure 5. There were three protein peaks. The middle peak appeared to be the inhibitor. The fast moving protein had a very interesting property; it was not precipitated with 5 per cent phosphotungstic acid. Tubes number 7-10 were pooled and lyophilized. The recovered inhibitor was 1,396 pg CT. Ultracentrifugal Studies The inhibitor fractions were subjected to study by ultracentrifugation. 1. Inhibitor treated with TCA— 12 mg of the material having specific activity of 494 were dissolved in 1 ml of citrate buffer (0,05 M citrate + 0.1 M NaCl, pH 3.0) and the solution was dialyzed against a O Figure 5.— Chromatography of a partially purified stable inhibitor in Sephadex G-100 gel filtration. Buffer used was 0.02 M Tris + 0.1 M NaCl, pH 7.7. against 500 ml of the citrate buffer overnight. A partition cell was used at a speed of 56,100 rpm. Two peaks appeartu on schlieren patterns. The fast sedimenting component had sedimentation coefficient ^s20 w^ 3*51 S and the slower sedimenting component had a value of 1.48 S. The fraction on top of the partition was taken after 165 minutes, and the sedi mentation coefficient was calculated on the basis of protein content and the inhibitor activity. The inhibitor was also ultracentrifuged at pH 7,7, 0.1 M Tris buffer. 2. Inhibitor fraction after Sephadex G-100— the lyophilized fraction was dissolved in 0.1 M Tris buffer (pH 7.7) and ultracentrifuged. The schlieren pattern indicated that there was only one peak, and the son was 1064 S. The sedimentation coefficient MV ^ VV was slightly lower than that calculated from the inhibitor activity. The s0„ calculated from the J 20 ,w inhibitor activity was 1.89 S and that from protein content was lo00 S. The schlieren pattern indicated that the gel filtration on Sephadex G-100 seemed to remove the fast sedimenting component. Yet, the very slow sedimenting component was left behind the inhibitor in the ultracentrifugation. These results are given in Tables VI and VII. 53 TABLE VI ULTRACENTRIFUGATION OF FRACTIONS FROM TCA TREATED AND SEPHADEX G-lOO AFTER TCA TREATMENT Samples Time in Minutes s c * * ... pi 20, w* S Sp2 Cell Inhibitor after TCA 120 1 o 72 2 o 99 Standard 0.1 M Tris pH 7.7 180 1.53 2.99 Inhibitor 120 1.48 4.16 after TCA 140 1 o 34 3.51 Part ition 0.05 M citrate pH 3.0 160 1.29 Inhibitor 64 1.60 0 after Sephadex 96 1.65 0 0.02 M Tris 128 1 o 66 0 Standard + 0.1 M NaCl 160 1.64 o pH 7.7 192 1.65 0 •Sedimentation coefficient calculated from schlieren patterns. **s values calculated for the slower sedimenting component. ***s values calculated for the fast sedimenting component. 54 TABLE VII ULTRACENTRIFUGATION USING PARTITION CELL Partition Cell Time in Minutes OD 280 mp* pg CT/ml of Diluted Sample Specific Activity pg CT/OD 280 mp Origin .283 140.0 495 Top 165 .187 55.5 297 Bottom .429 316.0 738 s Calculated from Activity 1.89 x 10“13 s Calculated from Protein Content 1.00 x 10"13 ♦Samples were diluted to fifteen times with saline. Note: Inhibitor fraction from TCA extract was used. Sedimentation coefficients (s) were calculated from the inhibitor activity and from protein content. The solvent was 0.05 M citrate buffer + 0.1 M NaCl, pH 3.0. 55 Paper Electrophoresis Serum and serum fractions obtained during the purification process were subjected to paper electro phoresis. The trypsin inhibitor in eluates from the paper was measured. Inhibitor activity in serum and DEAE-I was found with the «'1-globulin. DEAE-II fraction showed widespread inhibitor activity through the c^-globulin and albumin regions. The TCA-treated sample had two distinct peaks: one corresponding to albumin, and another slower moving inhibitor identi fied in the region of ^-globulin. These results are shown in Figure 6 and Tables VIII and IX. Vertical Column Electrophoresis Serum and a fraction from DEAE-II were run on vertical column electrophoresis. The results are shown in Figures 7 and 8. The active fraction obtained from the Sephadex G-100 column was also studied by vertical column electrophoresis since the analysis by paper electro phoresis had shown two inhibitors. Une migrated in the region of albumin, and the other remained in the region of -globulin. The fraction, having specific activity 455 and containing 2,340 pg CT, was applied on the column. Three ml fractions per three minutes 56 Figure 6.— Paper electrophoretic patterns. The technique described in the text was used. 1— Plasma, 2— A fraction from DEAE-I column, 3— A fraction from DEAE-II column, 4— A fraction from TCA extract. Two inhibitor peaks were recovered in sample number 4. 57 TABLE VIII PAPER ELECTROPHORESIS OF INHIBITORS FROM PLASMA, DEAE-I AND II, AND TCA EXTRACT Segment UK CT/ml Eluate Number Plasma DEAE-I DEAE-II TCA 1 0 0 0 8 2 0 0 3 14 3 8 8 10 6 4 14 14 14 5 5 11 14 14 9 6 0 6 14 14 7 0 o 0 14 8 0 0 0 0 i ¥ ' A u-:1 ' - r - ' i ^ i > 1 Segment Number Note: Paper electrophoresis at pH 8.65, 0.1 M barbital buffer. Segments were cut according to the protein peaks and the proteins were eluted with 1.0 ml 0.17 M Tris +0.01 M CaCl2, pH 7.7. The inhibitor content was determined in each segment. Plasma, DEAE-I, and DEAE-II had one inhibitor peak. TCA extract had two inhibitor peaks. 58 TABLE IX PAPER ELECTROPHORESIS OF FRACTIONS OBTAINED FROM SEPHADEX G-lOO, TCA, AND DEAE-II Segment Number Vig CT/ml Eluate Sephadex G-lOO TCA DEAE-II 1 o o 0 2 o 0 0 3 720 20 0 4 11 69 25 5 14 29 18 6 14 14 10 7 0 41 o 8 0 0 o 9 0 0 0 Segment Number £ 4 " 1 & £ 8 59 Figure 7.— Vertical column electrophoresis of serum (1.5 ml). A technique described in the text was used. 60 Figure 8.— Vertical column electrophoresis of DEAE-II fraction. The technique described in the text was used. 61 were collected. Two electrophoretically distinct proteins were obtained. The fast moving fraction was collected in tubes number 11-19 and the slower moving fraction was in tubes number 24-34. The fast moving fraction had specific activity 622, and 518 pg CT were obtainedc The slower moving component had specific activity average of 512, and 759 pg CT were recovered. The total recovery of the inhibitor was 55 per cent of the inhibitor applied on the column electrophoresis. Both fractions were dialyzed and lyophilized. The results are shown in Figure 9» Gel Filtration The inhibitor fraction from Dn,AE-II, 150 mg, was filtered through Sephadex G-100 in buffer system, 0.02 M Tris + O'.l M NaCl, pH 7.7. The bed of the column was 2.8 x 100 cm, and the flow rate was 3 ml per fifteen minutes at room temperature. Two protein peaks appeared as well as two inhibitors, as shown in Figure 10. Tubes number 50-59 were pooled to give fraction I and tubes number 64-70 to give fraction II. Both fractions were shown to be stable inhibitors and had electrophoretic mobilities similar to the inhibitor from DEAE-II. The analysis of the paper electrophoresis showed that the slower moving stable inhibitor was 62 Figure 9.— Vertical column electrophoresis of the stable inhibitor fraction from Sephadex G-100 gel filtration after TCA treatment. The technique described in the text was used. 63 - V / \j ] i, M t . i f- t t Figure 10.— Sephadex G-100 gel filtration. Inhibitor fraction obtained from DEAE-II was applied to a 2.8 x 100 cm column. Buffer used was 0.02 M Tris + 0.1 M NaCl, pH 7.7. 64 found when a large quantity of the inhibitor fraction was applied. Since both pooled fractions had equal electrophoretic properties, it is likely that the two inhibitor peaks were the same inhibitor. The results are shown in Table X. Effect of TCA and Zinc Ion on the Stable Inhibitor The appearance of two distinct stable inhibitors on paper electrophoresis after the treatment with TCA led to the investigation of the effect of zinc ion and TCA, or TCA alone, on the stable inhibitor. The stable inhibitor from fraction I of gel filtration was treated with Zn-TCA and TCA alqne, but without separating the precipitates from the super natants. The mixtures were dialyzed and lyophilized, and subsequently analyzed by paper electrophoresis. The results shown in Table XI were identical with those obtained with the untreated fractions (Table X). Identification of the Stable Inhibitor Starch Electrophoresis The electrophoretic patterns of trypsin inhibitors and the trypsin-binding protein in serum were studied. 65 TABLE X PAPER ELECTROPHORESIS OF FRACTIONS OBTAINED FROM SEPHADEX G-lOO AFTER DEAE-II COLUMN Segment Number Uff CT/ml Eluate Fraction I Fraction II 1 0 - 0 - 2 5.2 - o - 3 15.0 - 6.1 5 4 8.8 9.2 o o 5 17.8 19.2 0 o 6 >14 140 >14 80 7 >14 91 >14 64 8 0 o o o Segment Number * 1 . > '• V ’ l ., f - 2 Pm Cn y s s s ' s " 1 S " s S' , ' s ^ s ^ s ' ^ \ s s ' ^ ■ 1 - V I 6 8 Note: The technique described in the text was used. The results of duplicates of each fraction are given. Two inhibitors were recovered. The amount of slower moving component was less than that of the fast moving one. 66 TABLE XI ANALYSIS OF THE Zn-TCA AND TCA TREATED STABLE INHIBITOR ON PAPER ELECTROPHORESIS Segment Number pg CT/ml Eluate Zn-TCA Treated TCA Treated 1 o 0 o 0 2 2 - 5 6 3 10 13 18 16 4 6 4 4 4 5 >14 27 >14 25 6 >14 108 >14 140 7 8 5 >14 25 8 o 0 o 0 Segment Number P7-V C " \ -\ c r . .1 t K. r" ' I XL b'l :

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Creator Park, Mai Young, 1934- (author)

Core Title Purification and identification of trypsin inhibitors in human serum

School Graduate School

Degree Doctor of Philosophy

Degree Program Biochemistry

Degree Conferral Date 1964-06

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Mehl, John W. (committee chair), Haverback, Bernard J. (committee member), Marx, Walter (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-345476

Unique identifier UC11359186

Identifier 6412459.pdf (filename),usctheses-c18-345476 (legacy record id)

Legacy Identifier 6412459.pdf

Dmrecord 345476

Document Type Dissertation

Rights Park, Mai Young

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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