Further purification and characterization of acid-stable protease inhibitor from ascites of an ovarian carcinoma patient

Clinica Chimica Acta, 142 (1984) 47-60 Elsevier

47

CCA 02940

Further purification and characterization of acid-stable protease inhibitor from ascites of an ovarian carcinoma patient Kenji Akazawa a**, Hiroyuki Sumi a, Masugi Maruyama a and Hisashi Mihara b a Department of Physiology, Miyazaki Medical College, 5200 Kihara, Kiyotake -rho, Miyaraki - gun, Miyazaki - ken 889 - 16 (Japan) and b Umeh University, 901- 85 Umed (Sweden) (Received January 3rd, 1984; revision May 17th, 1984)

Key words: Acid-stable

protease inhibitor; Urinary ttypsin inhibitor; Ovarian carcinoma; Ascites

suuuualy An acid-stable protease inhibitor (AS-PI) has been previously demonstrated in ascitic fluid from patients with ovarian carcinoma. In this study, the AS-PI was further purified using DEAE-cellulose and isoelectric focusing (IEF), and a partial characterization was undertaken. On DEAE-cellulose ion-exchange column chromatography, AS-PI activity was observed in both adsorbed and non-adsorbed fractions. The former represented the main AS-PI peak. By IEF, the respective p1 values were 1.6 and 4.5. By gel filtration, the molecular weight of the main (adsorbed fraction) AS-PI was 78000. This AS-PI strongly inhibited trypsin and to a lesser extent chymotrypsin, but exerted no inhibitory effect on plasmin. It slightly inhibited SH proteases such as papain and ficin. Immunologically, AS-PI was distinct from cw,-antitrypsin, a,-antichymotrypsin, inter-cr-trypsin inhibitor, antithrombin III, C,-inactivator, cy,-macroglobulin and cu,-plasmin inhibitor. The main AS-PI reacted with and was neutralized by antiurinary trypsin inhibitor serum, and on immunoelectrophoresis, had a mobility slightly cathodal to serum albumin.

* Present address and reprint requests: Dr. Kenji Akazawa, Department of Gynecology, Tokyo Metropolitan Geriatric Hospital, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173, Japan. 0009-8981/84/$03.00

0 1984 Elsevier Science Publishers B.V.

48

Introduction Acid-stable protease inhibitor (AS-PI) was first reported in the urine by Muller in 1908 [l], and it was termed urinary trypsin inhibitor (UTI). Many investigators have since examined its physicochemical properties. Excretion of UT1 is known to increase in various physiological and pathological states, such as pregnancy, acute infection, advanced tuberculosis, rheumatic fever, nephritis and cancer [2-111. UT1 is a glycoprotein of high stability and solubility under acidic conditions [12]. Many investigators have discussed the origin of UTI. Proksch et al [13,14] and Hochstrasser et al [12] suggested a relation to plasma inter-a-trypsin inhibitor (ILuTI). However, more recently, Barthelemy-Clavey et al [15], Tanaka et al [16] and Akazawa et al [17] were unable to demonstrate any relation between highly purified UT1 and anti-IcwTI. Sumi et al [l&19] reported that the native type of UT1 (UTI-I, molecular weight (M,) 67 000) was readily transformed enzymatically to lower molecular forms of M, 45 000 (UTI-II) and 23 000 (UTI-III) under acidic conditions in urine, and to a form of M, 6000 by trypsin and papain treatments [20]. UT1 is a broad-spectrum protease which can inhibit trypsin [7,8,12,14,15,19,21], chymotrypsin inhibitor, [12,14,15,19,21], elastase [19], plasmin [19,21] and human acrosomal protease, acrosin [22,23]. Our previous report [17] demonstrated the presence of AS-PI in the tumor fluid and ascites of ovarian carcinoma patients. The AS-PI activity was higher in the tumor fluid. The AS-PI fractionated from the ascites was immunologically indistinguishable from UTI. The inhibitor migrated in the normal human serum albumin fraction on immunoelectrophoresis, and its M, was estimated to be 70000-80000 by gel filtration. In the present study, we carried out further purification and characterization of the AS-PI from the ascites of an ovarian carcinoma patient. Materials and methods An ascites sample was obtained from a 37-year-old patient with ovarian carcinoma (histologically mutinous cystadenocarcinoma). The ascites was taken at laparotomy, and no anti-cancer treatment had been given. Immediately after sampling, the ascites was stored at - 80°C until use. The unfrozen ascites (480 ml) was centrifuged at 2700 X g for 10 min, and 30% perchloric acid was added to the supernatant to give a final concentration of 6.0%. After standing for 1 h at room temperature, the acid-treated ascites was centrifuged at 1500 X g for 10 min at 4OC and then filtrated with a Whatman No. 50 filter paper. The eluate (450 ml) was dialyzed and lyophilized. The following substances were used: trypsin (type II), a-chymotrypsin and ficin (Sigma Chemical Co., St. Louis, MO, USA), human plasmin (Green Cross Corp., Osaka, Japan), papain (P-L Biochemicals Inc., Milwaukee, WI, USA), casein (Hammarsten) and agarose II (Wako Pure Chemical Indust. Co.), Sephadex G-100 (Pharmacia Fine Chemicals, Stockholm, Sweden), DEAE-cellulose (Seikagaku Kogyo Co., Osaka, Japan), bovine serum albumin and a-chymotrypsinogen (Sigma), egg

49

albumin (Boehringer Mannheim, FRG), rabbit anti sera against human whole serum, IaTI, cr,-antitrypsin (a,-AT), antithrombin III (AT-III), C,-inactivator (C,inact) and cY,-macroglobulin (a,-MG) (Behringwerke, Marburg, FRG), CY,-antichymotrypsin (Dako Immunoglobulin Ltd., Hostrup, Denmark), a,-plasmin inhibitor ((Y~-PI) (Mochida Pharmaceutical Co., Tokyo, Japan). All other chemicals were obtained from commercial sources and were of the best grade available. The native molecular form of UTI, UTI-I, was highly purified from normal human urine as described previously [18,19,21,24,25]. Anti-UTI-I serum was also obtained from rabbit serum as described previously [17]. Determination of anti-ttypsin activity The anti-trypsin activity was assayed by measurement of the ability to reduce the caseinolytic activity of trypsin as described previously [17,19]. One unit of AS-PI was defined as the amount inhibiting 1.0 pg of trypsin. Protein concentration was determined by the method of Lowry et al [26] using bovine serum albumin as a standard. Procedure of partial purification Partial purification of the acid-treated ascites was carried out in the sequence: gel filtration, DEAE-cellulose ion-exchange column chromatography, isoelectric focusing (IEF), and gel filtration. The details were as follows. Step 1. Gel filtration The lyophilized acid-treated sample was dissolved in 0.1 mol/l phosphate buffer (PB) containing 0.2 mol/l NaCl, pH 7.4, and was applied to a column of Sephadex G-100 (1.0 X 90 cm) previously equilibrated with the same buffer. The sample was eluted with the same buffer at 4OC, and 2 ml fractions were collected. Step 2. DEAE-cellulose ion-exchange column chromatography The active fraction obtained by gel filtration was pooled and dialyzed against 0.05 mol/l PB containing 0.05 mol/l NaCl, pH 7.4, for 24 h at 4OC, and applied to a DEAE-cellulose column (1.4 x 27 cm) previously equilibrated with the same buffer at room temperature. The column was eluted until the non-adsorbed protein had been almost completely eluted, and it was then eluted with a linear salt gradient to 0.5 mol/l NaCl using two 250 ml chambers at room temperature. Five ml fractions were collected. Step 3. Isoelectric focusing Each of the active fractions detected in the adsorbed and non-adsorbed materials by DEAE-cellulose ion-exchange column chromatography was collected and concentrated with polyethyleneglycol20 000 (PEG). Each was applied to an IEF column. The IEF was performed by the method of Vesterberg and Svensson [27] using carrier ampholytes of pH 3.5 to 10.5. The column (LKB 8100, 110 ml) was maintained at 4°C and the potential at 900 V for 36 h. Two ml of fractions were collected. Step 4. Gel filtration The active fraction collected by IEF from the DEAE-cellulose-adsorbed fraction was lyophilized and dissolved in 0.1 mol/l PB containing

50

0.2 mol/l NaCl, column prepared were collected.

pH 7.4. The resultant solution was applied to a Sephadex G-100 in the same way as for the first gel filtration. Two ml fractions

Immunological experiments Double immunodiffusion with rabbit anti-UTI-I serum was carried out by the method of Ouchterlony [28] at room temperature for 24 h. The gel concentration in Verona1 buffer (I = 0.07, pH 8.6) was 1.0%. Immunoelectrophoresis was performed with 1% agarose in Verona1 buffer (I = 0.07, pH 8.6) at 3 mA/cm for 90 min with cooling. The gel was stained with amido black. Neutralization effect of antisera against UTI-I and certain serum protease inhibitors on the AS-PI activity Various concentrations (in 100 ~1) of antisera against UTI-I, a,-AT, cr,-antichymotrypsin, IaTI, AT-III, C,-inact, (r,-MG or cy,-PI were mixed with a certain concentration and amount of fractions which had AS-PI activity after IEF. Following preincubation at 37’C for 30 min, an equivalent amount of trypsin (2 pg/20 ~1) was added and further preincubation was carried out at 37’C for 5 min. Casein solution (8%, 250 ~1) was then added, and the mixture was incubated at 37OC for 30 min. The neutralization effect was calculated from the residual caseinolytic activity, which was determined as described above. Determination of the inhibitory activity to several proteases The active fraction detected by IEF of the DEAE-cellulose-adsorbed active fraction was examined for its spectrum of inhibitory activity towards trypsin, chymotrypsin, plasmin, papain and ficin. The assay system used was as follows. Trypsin (1 pg/lOO pl), chymotrypsin (1 pg/lOO pl), plasmin (13.3 pg/lOO ~1 = 0.2 U/100 PI), papain (2 pg/lOO ~1) or ficin (1 pg/lOO ~1) was mixed with a certain amount of the active fraction and 0.1 mol/l PB containing 0.1 mol/l NaCl, pH 7.4, to a final volume of 750 ~1 (in the assay for papain and ficin, 5 PM cysteine was included) and preincubated at 37’ C for 5 min. After the preincubation, 250 ~1 of 8% casein solution was added and the mixture was incubated at 37OC for 30 min. After the addition of 10% perchloric acid to the mixture, the amount of tyrosine-like substance produced was determined by Lowry’s method [26]. The amount of these proteases in the assays was determined on the basis of the preliminary finding that the OD,,, in the caseinolysis of these proteases was between 0.5 and 0.6. Results

Gel-filtration The acid-treated ascites (450 ml; AS-PI activity, 8.42 U/ml) was lyophilized and dissolved in 8 ml of 0.1 mol/l PB containing 0.2 mol/l NaCl, pH 7.4, to give 37.5 mg/ml (AS-PI activity, 473.9 U/ml). Then, 0.4 ml of this solution was applied to the Sephadex G-100 column. As shown in Fig. 1, two inhibitor peaks were detected, which did not correspond to the protein peaks. The M, values of the two active

51

&A .2-

EA 1

aCTG J

Ft.0

1

1

5

10

15

20

25

30

35

40

45

50

Fig. 1. Gel-filtration on Sephadex G-100. Column, 1.0 x 90 cm; elution buffer, 0.1 mol/l phosphate buffer containing 0.2 mol/l NaCl, pH 7.4; flow rate, 5.8 mI/h; fraction volume, 2.0 ml. Applied sample: protein concentration, 37.5 mg/ml; volume, 0.4 ml, activity, 190 U. 0, I~bito~ activity; l , protein concentration (OD,,); , pooled fractions; BSA, bovine serum albumin; EA, egg albumin; aCTG, a-chymotrypsinogen.

I

1

*

10

$1

20

30

*

I

*

I,

40

50

60

70

20 Tuba

/

I

I,

so

100

110

I,

120

130

14(I

number

Fig. 2. Ion exchange cohmm c~mato~aphy on DEAE-ceBulose. Column, 1.4 X 27 cm; etution buffer, 0.05 mol/l PB containing 0.05 mol/l NaCl, pH 7.4 (fractions no. l-50); gradiented from fraction no. 51; flow rate, 52 ml/h; fraction volume, 5.0 ml. Applied sample: protein concentration, 2.55 mg/mf; vohune, 47 ntl, activity, 2054 U. 0, Inhibitory activity; 0, protein concentration (OD,,); - - - - - -, conductivity (mmho); -, pooled fractions.

52

peaks as estimated from the standard proteins were 78 000 and 52 000, respectively. The same procedure was repeated several times until the total volume of the applied sample attained 6.4 ml. DEAE-cellulose ion-exchange column chromatography Next, 47 ml of the 50 ml dialyzed active fraction obtained from gel filtration was applied to a DEAE-cellulose column. As illustrated in Fig. 2, two active peaks were detected, one in the non-adsorbed and the other in the adsorbed fraction. The AS-PI activity in the non-adsorbed fraction (fractions no. 9-28) was 198 U and the spec act was 6.3 U/mg protein. The AS-PI activity in the adsorbed fraction (fractions no. 77-128) was 1457.9 U and the spec act was 121.9 U/mg protein. The conductivity of the adsorbed fraction showing peak activity was found to be about 40 mmho using a conductivity meter (M&S Instruments Inc., Japan). Since a much higher activity was observed in the adsorbed fraction as compared to the non-adsorbed fraction, the AS-PI in the adsorbed fraction was termed the ‘main AS-PI’, and the AS-PI in the non-adsorbed fraction the ‘minor AS-PI’. Isoelectric focusing IEF of the active fractions in both the adsorbed and non-adsorbed fractions was carried out. Adsorbed fraction The active fraction (260 ml; fractions no. 77-128) of the adsorbed fraction was concentrated to 6.3 ml with PEG. This sample was dialyzed against distilled water for 1 h at 4OC to remove PB. Then, 5.0 ml of the 6.3 ml concentrate was applied to the IEF column. Fig. 3A shows the elution pattern of the adsorbed fraction. The main active peak was observed at p1 1.6. The spec act of the peak fraction (fractions no. l-6) was 289.8 U/mg protein. The recovery rate of total activity was 87.1% of the applied sample. Non-a&orbed fraction The active fraction (100 ml; fractions no. 9-28) of the non-adsorbed fraction was concentrated to 6.2 ml This sample was dialyzed in the same manner as for the adsorbed fraction, and 6.0 ml of the 6.2 ml concentrate was applied to the IEF column. Fig. 3B shows the elution pattern of the non-adsorbed active fraction. The active fraction was observed at p1 4.5, and the spec act of this peak fraction was 10.1 U/mg protein. The recovery rate of total activity was 57.5% of the applied sample. Subsequent experiments concerning the characterization of the main and/or minor AS-PI were carried out with the active fractions detected by IEF. Gel filtration The main AS-PI fraction was subjected again to gel filtration. That is, 19 ml of the main AS-PI fraction was lyophilized and dissolved in 2.0 ml of 0.1 mol/l PB containing 0.2 mol/l Nadl, pH 7.4, and 0.5 ml of this solution (total activity, 200 U; protein concentration, 1.38 mg/ml) was applied to a Sephadex G-100 column. As

53

shown in Fig. 4, one active peak was detected, and the M, of this peak was 78000. The recovery rates calculated from 300 mg of sample for each purification step are listed in Table I: 31.3-fold purification was achieved with a recovery of 38.8% of the activity of the acid-treated ascites. A

rl*

1001

6

I P

6

4

50

40

30 Tube

56

number

6 r, 6

I-

t

1

n 10

20

50

40

30 Tube

60

number

Fig. 3. Isoelectric focusing patterns. A. Adsorbed fraction from DEAE-cellulose. B. Non-adsorbed fraction from DEAE-cellulose. Column (LKB 8100), 110 ml; carrier ampholytes, pH 3.5-10.5; potential, 900 V; fraction volume, 2.0 ml. Applied samples: AS-PI activity - A: 1157 U; B: 181 U; protein concentration - A: 1.90 mg/ml; B: 4.4 mg/ml; volume - A: 5.0 ml; B: 6.0 ml. 0, Inhibitory activity; 0, protein concentration (OD,,); - - - - - -, pH; -, pooled fractions.

54

Immunological experiments On double immunodiffusion (Fig. 5) two precipitin lines were observed between anti-UTI-I and UTI-I containing its fragment. Two precipitin lines were also observed between anti-UTI-I and the main AS-PI fraction, and these fused completely with the corresponding lines involving UTI-I containing its fragment. However, no precipitin line was noted between anti-UTI-I and the minor AS-PI fraction. On double immunodiffusion, neither the main AS-PI nor minor AS-PI showed any precipitin line against anti-IarTI. On immunoelectrophoresis (not shown), the acid-treated ascites from the first

BSA EA

aCTG 1

:.:.:.:.:.: :::::. ,.:.:.:.:.:. ;;::::, :::;::. ,‘.‘.‘_‘.~.‘. :;;:::, ::::::_ ,‘.‘.‘.‘.‘.~. :::;::_

1

,

5

10

15

20

25

30 35 Tube number

40

45

50

Fig. 4. Gel filtration on Sephadex G-100. Applied sample: main AS-PI fraction, protein concentration, 1.38 mg/ml; volume, 0.5 ml; activity, 200 U. Other details as Fig. 1

TABLE I Purification

of the main AS-PI from ascites of an ovarian carcinoma

Fraction

Acid treatment Gel filtration DEAJkellulose Isoelectric focusing Gel filtration

patient Yield

Degree of purification (fold)

Total protein

AS-PI activity

SPec act

(mg)

(U)

(U/mg)

308.0 159.4 15.9

3791.2 2731.3 1938.7

12.6 17.1 121.9

100 72.0 51.1

1.00 1.36 9.68

4.6 3.7

1340.4 1469.1

289.8 395.8

35.4 38.8

23.00 31.30

(X)

55

Fig. 5. Double immunodiffusion against anti-UTI-I serum. Center well: rabbit anti-UTI-I serum. Peripheral wells: 1, 4 (0.3). UTI-I containing its fragment; 2, 5 (0.145), main AS-PI fraction; 3, 6 (0.59), minor AS-PI fraction. Numbers in parentheses are for protein concentration in mg/ml. 10 pl in each well.

A

B

loo-

100

00-

00

60-

60

40-

40.

zo-

20

n-

l-i-

0

0

I+9

6

1

6

5

Concentration

d-

0

0

96765 of

anti

UTI-I

90165

C-log)

Fig. 6. Neutralization effect of anti-UTI-I on AS-PI activity. AS-PI fraction. Ordinate, residual AS-PI activity; abscissa, Absence of anti-UTI-I.

A. UTI-I. B. Main AS-PI fraction. C. Minor anti-UTI-I serum concentration (-log). 0,

56

step of purification fused with anti-UTI-I serum at the position of the arc of human serum albumin. The main AS-PI fused with anti-UTI-I serum at a cationic position with respect to n,ormal human serum albumin. Each precipitin lines showed double arcs. However, the minor AS-PI exhibited against anti-UTI-I serum.

normal slightly of the no arc

Neutralization effect of anti-UTI-I on the AS-PI activity As shown in Fig. 6, each inhibitory activity in the main AS-PI and UTI-I was neutralized almost completely by anti-UTI-I serum. However, the AS-PI activity in the minor AS-PI fraction was not neutralized at all. The main and minor AS-PIs were not neutralized by antisera against the well-known serum protease inhibitors, cr,-AT, cu,-antichymotrypsin, I~YTI, AT-III, C,-inact, LX,-MG and.q-PI. Determination of the inhibitory activity towarak several proteases As shown in Fig. 7, 50% inhibition of 1 pg of trypsin and chymotrypsin by the main AS-PI fraction was calculated to occur at 2.76 and 30.36 pg, respectively.

x

100 -

SO-

“1L

D .

50

I

o-

OJ r

0

10

1

2out

% 150

100

1

Fig. 7. Inhibitory activity of main AS-PI fraction towards proteases. A. Trypsin. B. Chymotrypsin. C. Plasmin. D. Papain. E. Ficin. Ordinate, residual caseinolytic activity; abscissa, amount of active ads. fraction (0.145 pg/pl).

57

However, no inhibition was detected with and/or ficin was detected, but 50% inhibition

plasmin. Slight inhibition could not be attained.

of papain

Discussion

In a previous study [17], we found AS-PI in the ascites and tumor fluid of patients with ovarian tumor. A much higher AS-PI activity was observed in the ascites and tumor fluid of patients with malignant ovarian tumors than in those of patients with benign tumors. We also reported that the AS-PI in the ascites of malignant ovarian tumors showed a M, of 70000-80000 by gel filtration. The AS-PI migrated in the human serum albumin fraction on immunoelectrophoresis. The AS-PI showed reactivity to anti-UTI-I in double immunodiffusion and neutralization tests. In the present study, we carried out further purification of AS-PI from the ascites of a patient with ovarian carcinoma and achieved a partial characterization including the isoelectric point and inhibitory spectrum. We detected two types of AS-PI in the ascites: one, the main AS-PI, reacted with anti-UTI-I, whereas the other, the minor AS-PI, did not. The M, and antigenicity of the main AS-PI in the ascites to anti-UTI-I corresponded to those of the acid-treated ascites previously reported. The M, of the main AS-PI was estimated to be 78000 by gel filtration. This value was rather higher than that of UTI-I (67000). On double immunodiffusion against anti-UTI-I serum, two precipitin lines were detected with the main AS-PI fraction, and each line fused to the corresponding precipitin line of UTI-I containing its fragment. Since the main AS-PI fraction had one active peak on gel filtration (step 4), the inner thin precipitin line was probably formed by the component which showed no inhibitory activity in spite of its reactivity to anti-UTI-I. On immunoelectrophoresis, the main AS-PI fraction migrated to a slightly cationic position with respect to human serum albumin. This result differed slightly from our previous report that the AS-PI migrated in the position of human serum albumin. Sumi et al [19] found that highly purified UTI-I migrated in the position of prealbumin. The migration position of the AS-PI in ascites may possibly shift nearer to the position of prealbumin from albumin with purification. Concerning the neutralization effect with anti-UTI-I, the inhibitory activity of the main AS-PI fraction was almost completely neutralized as for purified UTI-I. In our previous study, the AS-PI activity of acid-treated ascites was not completely neutralized, but about 35% of the AS-PI activity remained. Since the minor AS-PI which had no reactivity to anti-UTI-I was removed from the AS-PIs in the acid-treated ascites by DEAE-cellulose ion-exchange chromatography, the main AS-PI which had reactivity to anti-UTI-I should show complete neutralization by anti-UTI-I. The AS-PI not neutralized by anti-UTI-I in the previous report, presumably corresponds to the minor AS-PI. In the tests of the neutralization effect with antisera against well-known serum protease inhibitors, the AS-PI activity of the main and minor AS-PI fractions was not neutralized at all. This result suggests that neither the main nor minor AS-PI was identical to well-known serum protease inhibitors.

58

TABLE II Comparative

properties of UTI-I and AS-PI prepared from ascites UTI-I

AS-PI in ascites

M, by gel filtration Isoelectric point Inhibitory spectrum Trypsin Chymotrypsin Plasmin Ficin Papain Antigenicity against Anti-UTI-I Anti-IuTI

67000 * 2.0 **

78000 1.6

(+++)**

(++I (+) (-) (k) (It)

Position on immunoelectrophoresis

prealbumin *

References:

*(19], **[20], ***[25],

(+I ** (zk) ** (-)

***

(_)***

(+I (-)

***+

(+) (-) slightly to the cationic side of albumin

****[15-171.

The isoelectric point of the main AS-PI was 1.6. This AS-PI inhibited trypsin activity most strongly and chymotrypsin to a lesser extent, but did not inhibit plasmin activity. Sumi et al [20] reported the following results for the p1 and inhibitory spectra of UTI-I. The p1 of UTI-I was 2.0, and that of UTI-I fragment was shifted to a more alkaline position on fragmentation by papain. UTI-I inhibited trypsin activity most strongly followed by chymotrypsin and plasmin. The inhibitory spectrum was modified after fragmentation: the inhibitory activity towards trypsin was reduced, and that towards plasmin rose. The p1 and inhibitory spectrum of the main AS-PI in the present study were very similar to those of purified UTI-I, but did not correspond completely. Comparison of our data with those given by Sumi et al revealed that the p1 of the main AS-PI was more acidic than that of UTI-I and the ratio of anti-trypsin activity to anti-chymotrypsin activity for the main AS-PI was higher than that of UTI-I. The characteristics of the main AS-PI in ascites are summarized in Table II in comparison with those of UTI-I. From the present results, it is suggested that the main AS-PI in the ascites of ovarian carcinoma patients may represent one of the precursors of UTI. Various problems concerning the pathophysiological significance of AS-PI in the ascites of ovarian carcinoma patients still remain, and further work is needed to characterize this inhibitor fully. Acknowledgements I wish to express my hearty gratitude to Mr. A.J. Smith for correcting the manuscript. This work was supported in part by a grant (No. 58771080) for scientific research from the Ministry of Education, Science and Culture of Japan.

59

References 1 Miiller E. Uber das Verhalten des proteolytischen Leucocytenfermentes und seines ‘Antifermentes’ in den normalen und krankhaften Ausscheidungen des menschlichen Kdrpers. Dtsch Arch Klin Med 1908; 92: 199-216. 2 Schippers JC. Uber die antitryptische Wirkung pathologischer Harne. Arch Klin Med 1911; 101: 543-556. 3 Fujimoto B. Studies on the antitrypsin of serum. J lmmunol 1918; 3: 51-66. 4 Dillard GHL. The trypsin inhibitor of the urine in health and disease. J Lab Clin Med 1950; 36: 266-211. 5 Faarvang HJ. Relation between pituitary-adrenal system and excretion of trypsin inhibitor in urine. Proc Sot Exp Biol Med 1958; 98: 89-91. 6 Faarvang HJ. The excretion of trypsin inhibitor in urine in normal persons. Acta Endocrinol 1959; 30: 285-295. 7 Mayehiro A. Studies of trypsin inhibitor in urine. II. The trypsin inhibitor of the urine in various pediatric patients. Yokohama Med Bull 1960; 11: 111-124. 8 Faarvang HJ. The urinary trypsin inhibitor in man (‘Mingin’). Stand J Clin Lab Invest 1965; 17: l-78. 9 Smith JM, Balabanian MB, Freeman RM. Serum levels of a component reacting with antiserum to urinary antitrypsin in health and disease with emphasis on high levels in renal failure. J Lab Clin Med 1976; 88: 904-913. 10 Toki N, Hayashi M, Maehara S, Sumi H. Studies on urinary trypsin inhibitor (UTI). A new determination method of UTI. Acta Urol Jpn 1978; 24: 1053-1060. 11 Toki N, Sumi H. Urinary trypsin inhibitor and urokinase activities in renal disease. Acta Haematol 1982; 67: 110-114. 12 Hochstrasser K, Bretzel G, Feuth H, Hilla W, Lempart K. The inter-a-trypsin inhibitor as precursor of the acid stable protease inhibitor in human serum and urine. Hoppe-Seyler’s Z Physiol Chem 1976; 357: 153-162. 13 Proksch GJ, Lane J, Nordschow CD. Interrelation of the urinary trypsin inhibitor to human plasma inter-alpha-trypsin inhibitor. Clin Biochem 1973; 6: 200-206. 14 Proksch GJ, Routh JI. The purification of the trypsin inhibitor from human pregnancy urine. J Lab Clin Med 1972; 79: 491-499. 15 Barthelemy-Clavey V, Yapo EA, Vanhoutte G, Hayem A, Mizon J. Purification et characttrisation des inhibiteurs de proteases de l’urine humaine. Biochim Biophys Acta 1979; 580: 154-165. 16 Tanaka Y, Maehara S, Sumi H, Toki N, Moriyama S, Sasaki K. Purification and partial characterization of two forms of urinary trypsin inhibitor. Biochim Biophys Acta 1982; 705: 192-199. 17 Akazawa K, Sumi H, Maruyama M, Mihara H. Acid stable protease inhibitor in ascites of ovarian carcinoma. Clin Chim Acta 1983; 131: 87-99. 18 Sumi H, Minakata K, Takada Y, Takada A. Trypsin inhibitors in human urine. J Physiol Sot Jpn 1977; 39: 53-58. 19 Sumi H, Takada Y, Takada A. Studies on human urinary trypsin inhibitor. 1. Its modification on treatment of urine with acid. Thromb Res 1977; 11: 747-754. 20 Sumi H, Toki N, Takasugi S et al. Low molecular weight trypsin-plasmin inhibitors isolated from papain-treated urinary trypsin inhibitor. Thromb Haemostas 1982; 47: 14-18. 21 Toki N, Maehara S, Hayashi M, Ukai R, Sumi H. Further purification and some properties of human urinary trypsin inhibitor. Invest Urol 1980; 17: 465-469. 22 Sumi H, Toki N. Inhibitors of acrosin and SH-proteinase in normal human urine. Proc Sot Exp Biol Med 1981; 167: 530-535. 23 Sumi H, Toki N. Inhibitors of acrosomal proteinase acrosin: human urinary trypsin inhibitor (UTI) and 4-(2-carboxyethyl) phenyl trans-4-aminomethylcyclohexanecarboxylate hydrochloride (DV-1006). Experientia 1980; 36: 1103-1104. 24 Cuatrecasas P. Protein purification by affinity chromatography. J Biol Chem 1970; 245: 3059-3065. 25 Sumi H, Maruyama M, Mihara H, Maehara S, Toki N. Human urinary trypsin inhibitors purified by affinity chromatography on trypsin-Sepharose. Acta Haematol Jpn 1980; 44: 146-153.

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26 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275. 27 Vesterberg 0, Svensson H. Isoelectric fractionation, analysis, and characterization of ampholytes in natural pH gradients. Acta Chem Stand 1966; 20: 820-827. 28 Ouchterlony 0. Diffusion in gel methods for immunological analysis. Prog Allergy 1958; 5: l-78.