Preparation and some properties of highly purified human serum kallikrein

321

Clinica Chimica Acta, 93 (1979) 321-327 0 Elsevier/North-Holland Biomedical Press

CCA 9987

PREPARATION AND SOME PROPERTIES SERUM KALLIKREIN

G.A. YAROVAYA M. KAWALEC b

OF HIGHLY

PURIFIED

HUMAN

a, V.L. DOTSENKO a, V.N. OREKHOVICH a, J. KAWIAK b and

a Chair of Biochemistry of the Central Institute for Advanced Training of Physicians, Moscow (U.S.S.R.) and b Medical Center of Post-graduate Education, Cytophysiological Laboratory, Warsaw (Poland) (Received August 31st, 1978)

summary A 3000-6000-fold purified kallikrein was obtained from human serum in lo-25% yield by chromatography on QAE-Sephadex A-50, Molselect CM-50 and on soybean trypsin inhibitor (SBTI)-AH-Sepharose 4-B. The enzyme had a specific activity of 14-23 U, as measured by BAEE hydrolysis. Some properties of highly purified kallikrein are described.

Introduction The main kinin-forming enzyme kallikrein occurs in human and mammalian plasma in exceedingly small amounts which complicates the process of purification and the study of properties of this enzyme. The currently available methods for purification of kallikrein from human [l-3], bovine [ 41, porcine [S] and rabbit [63 plasma are very laborious, and the preparations obtained by these methods possess low specific activity. The application of affinity chromatography proved to be highly effective, allowing a IlOO-l200-fold purification of serum kallikrein 173 ; it was employed at the second and the third steps of purification using soybean trypsin inhibitor (SBTI) and benzamidine which were immobilized on Sepharose 4-B. The present paper reports on the purification of kallikrein from human serum employing ion-exchange chromatography on QAE-Sephadex A-50 and Molselect CM-50, as well as affinity chromatography on SBTI coupled with AH-Sepharose 4-B. Some properties of the enzyme, possessing high e&erase and kininogenase activities, are described.

322

Materials and methods Reagents The following reagents were used: QAE-Sephadex A-50, AH-Sepharose 4-B, Sephadex G-200 (Pharmacia, Sweden); Molselect CM-50, bradykinin triacetate, N-benzoyl-L-arginine ethyl ester (BAEE) (Rear& Hungary); 4-ethyl-3-dimethylaminopropyl carbodiimide, tosyllysyl chloromethylketone (TLCK) (Serva, F.R.G.); Trasylol (Bayer, F.R.G.); Gordox (Gideon Richter, Hungary) Kontrical (VEB, D.D.R.); trypsin (Spofa, Czechoslovakia); ovomucoid, Pantripin (U.S.S.R.); p-tosyl-L-arginine methyl ester - HCI (TAME), soybean trypsin inhibitor (SBTI) Type II S (Sigma, U.S.A.), N-tosyl-L-lysine methyl ester, hydrochloric (TLME) (Calbiochem, U.S.A.). Serum was obtained from fresh donor blood. E&erase activity of kallikrein was determined by BAEE hydrolysis followed by changes in absorbance at 253 nm and 25°C; it was expressed as pmol or nmol of BAEE hydrolyzed per min, i.e. in U or mU respectively. The reaction mixture contained 0.1-0.2 ml of the enzyme, 0.1-0.2 ml of 0.1 M Tris-HCl buffer, pH 8.8, 1.8-1.6 ml of 0.05 M Tris-HCl buffer, pH 8.0 and 1 ml of 1.5 X 10e3 M BAEE solution. TAME-e&erase activity of kallikrein was determined by changes in absorbance at 247 nm and 25°C; the reaction mixture contained 0.1 ml of the enzyme, 0.1 ml of 0.1 M Tris-HCl buffer, pH 8.8, 2.2 ml of 0.05 M Tris-HCl buffer, pH 8.0, and 0.6 ml of 5.0 X 10T3 M TAME solution. TLME-esterase activity of the enzyme was determined by changes in absorbance at 248 nm and 25°C; the reaction mixture contained 0.1 ml of the enzyme, 0.1 ml of 0.1 M Tris-HCl buffer, pH 8.8, 2.6 ml of 0.05 M Tris-HCl buffer, pH 8.0, and 0.2 ml of 10.6 X 10v3 M TLME solution. Kininogenase activity of kallikrein was tested on the isolated estrus rat uterus which was stimulated by diethyl stilboesterol 24 h before the experiment. Human serum, heated for 1 h at 61°C and dialyzed overnight against saline, was used as substrate. The reaction mixture contained 1 ml of the substrate, 0.5 ml of 0.1 M Tris-HCl buffer, pH 7.5, containing 0.003 M 8-hydroxychinoline and 0.05 ml of the enzyme solution. After 1, 2 and 5 min, the reaction was stopped by adding 0.2 ml of 12.5% TCA; the pH of the mixture was adjusted to 7.0 by adding 0.5% NaOH, and the samples were tested on the isolated estrus rat uterus in a water bath containing Jalon solution (12 ml) at 30°C. Kallikrein activity was expressed in pg bradykinin liberated from human serum. The activation of kallikreinogen with trypsin was carried out for 15 min at 25°C. The samples contained kallikreinogen (10 ml), 0.1 M Tris-HCl buffer, pH 8.8 (2 ml) and trypsin (200 pg). At the end of the incubation, 6 mg of ovomucoid was added, and 10 min later aliquots of 0.1-0.2 ml were taken from the incubation mixture and tested for BAEE-esterase activity. Results and discussion At the first step, kallikrein was separated in the form of its precursor kallikreinogen from other serum proteinases in a 1.6 X 45 cm column of QAE-

323

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25 I 2.0 -

!.

1.5-

: mo 9”

IO-

0.5

r I’

----h&d30

Elutton

120 volume

b 150 (ml)

180

’

210

Fig. 1. Chromatography of kalliieinogen on M&elect CM-50. Kalliheinogen (90 mg) in 0.025 M Na phosphate buffer. pH 6.5. was applied to a 1.6 X 25 cm column of M&elect CM-50 and equiliberated with the same buffer. After washing the column with the original buffer, the proteins adsorbed were eluted with a linear gradient of O-O.3 M NaCl at a flow rate of 12 ml/h. Fractions of 3 ml each were collected. and the protein content and esterase activity in the fractions were determined.

Sephadex A-50 equilibrated with 0.1 Tris-HCl buffer, pH 8.0, containing 0.003 M EDTA. The column was loaded with 30-40 ml of serum diluted with the same buffer. The separation of serum proteins was carried out at room temperature and immediately after emerging from the column, the fractions were cooled. Kallikreinogen was found in the unadsorbed pooled protein fraction (volume 140-150 ml) with an A 280 of 500.-700 and a specific esterase activity of 20-30 mU after activation of the precursor. Kallikreinogen-containing fractions were collected and successively dialyzed against 0.0125 M sodium phosphate buffer, pH 6.5, at 4°C for 12 h and against 0.025 M sodium phosphate buffer at the same pH and temperature for 6 h. The next step involved chromatography on a 1.6 X 25 cm column of Molselect CM-50 equilibrated with 0.025 M sodium phosphate buffer, pH 6.5. After the material from the first step was applied to the column, it was washed with the original buffer, and the proteins adsorbed were eluted with a linear gradient of O--O.3 M NaCl by passing 200 ml of the gradient solution through the column. Kallikreinogen was found in the protein fractions eluted with 0.15-0.2 M NaCl (Fig. 1). The pooled kallikreinogen fraction (30-40 ml) had an Azso of 150-200 and a specific activity of 60--100 mU. The third step involved affinity chromatography on SBTI immobilized an AH-Sepharose 4-B. To prepare the immobilized enzyme inhibitor, 77 mg of SBTI was dissolved in 0.1 M phosphate buffer, pH 5.0, and dialyzed overnight against the same buffer. The dialyzed inhibitor (15 ml) and carbodiimide (300 mg) were added to about 10 ml (2.5 g) of AH-Sepharose 4-B. The suspension was allowed to stand overnight at 4°C with constant stirring, then it was successively washed

324

on a glass filter with 0.1 M phosphate buffer, pH 5.0, distilled water, 0.3 M KCl/HCl, pH 2.0, and again with phosphate buffer. The suspension of immobilized SBTI was stored in phosphate buffer, pH 5.0, to which sodium azide was added up to a final concentration of 0.02%. A l-ml amount of the precipitated suspension of immobilized inhibitor in 0.046 M Tris-HCl buffer, pH 8.1, containing 0.01 M CaCl, was found to bind 3.16 mg of trypsin (or 173 units of TAME-esterase activity). The immobilized SBTI thus obtained was used for further purification of kallikrein; 0.8 ml of SBTI-AH-Sepharose 4-B was mixed with 1.2 ml of Sephadex G-200 and applied to a 1 X 2 cm column previously washed with 0.05 M Tris-HCl buffer, pH 8.0. The kallikreinogen-containing fraction, prior to purification on SBTI-AHSepharose 4-B, was deslated either by filtration through a Sephadex G-25 column in 0.05 M Tris-HCI buffer, pH 8.0, or by dialysis against the same buffer. The proenzyme was activated by trypsin, the activity of the latter was suppressed by ovomucoid, the solution was passed through a column of immobilized SBTI at a flow rate of 0.25 ml/min, and 2-ml fractions were collected. After washing the column with 0.05 M Tris-HCl buffer, pH 8.0, the proteins bound to the inhibitor were successively eluted with 0.3 M NaCl in 0.05 M TrisHCl buffer, pH 8.0, and with 0.3 M NaCl/HCl, pH 3.5. The results of a typical experiment on purification of kallikrein by the above procedure are presented in Fig. 2. In this experiment, 112 ml of enzymic solution (14.5 U of enzymic activity) with a specific activity of 100 mU was passed through the column. Upon washing the column with 0.3 M NaCl in 0.05 M Tris-HCl buffer, pH 8.0, ballast proteins with traces of e&erase activity were

Fraction

number

(2 ml

each)

Fig. 2. Affinity chromatography of kallikrein on SBTI-AH-Sepharose 4-B. After activation of kallikreinogen, 112 ml of the enzyme solution (14.6 U of esterase activity) in 0.05 M Tris-HCl buffer, PH 8.0. was applied to a SBTI-AHSepharose 4-B column. The column was washed with the buffers indicated in the figure at a flow rate of 0.25 ml/min. Fractions of 2 ml each were collected, and the protein content and BAEEesterase activity in them, were determined.

325

eluted. Kallikrein was eluted with an acidic solution of 0.3 M NaCl. The specific activity of the enzyme in the peak fraction reached 29 U. As the solution passed through the SBTI-Sepharose 4-B column containing the trypsin-ovomucoid complex, it was necessary to study the behaviour of the latter in the column: indeed, the breakdown of the complex and binding of trypsin to SBTI could contaminate the kallikrein fraction with trypsin in the course of the elution. The stability of the complex in the presence of immobilized SBTI was checked by passing trypsin totally inhibited by ovomucoid through a SBTI-AHSepharose 4-B column (1 X 3 cm) washed with several volumes of 0.05 M TrisHCl buffer, pH 8.0. The complex was prepared in the course of a 10 min reaction at 25°C using 0.5 mg of trypsin and 10 mg of ovomucoid in 2.5 ml of 0.05 M Tris-HCl buffer, pH 8.0. Upon passing the trypsin-inhibitor complex through the column, it was successively washed with 0.05 M Tris-HCl buffer, pH 8.0, 0.3 M NaCl/HCI, pH 3.5, and 0.3 M KCl/HCl, pH 2.0. The fractions of 2 ml each were tested for BAEEesterase activity, and the absorbance at 280 nm was measured. No free trypsin was found in the eluates which was indicative of the stability of the complex. Thus, the preliminary purification of kallikreinogen and application of affinity chromatography on SBTI-Arm-Sepharose, enabled us to obtain 30006000-fold purified kallikrein preparations with a specific BAEE-esterase activity of 14-30 U and kininogenase activity corresponding to 36 pg bradykinin equivalents released from heated human plasma per min per unit of kallikrein-e&erase activity. The scheme of purification of kallikrein is given in Table I. The data of a typical experiment presented here show that affinity chromatography sharply increased the efficiency of purification: between the second and the third steps, the purification index by kininogenase activity was 3 times higher than that by e&erase activity. The kallikrein preparations obtained were stable for several days in 0.3 M NaCl/HCl buffer, pH 3.5, at 4°C. The activity was best retained at pH 5.06.5 and 4°C in the presence of 0.02% sodium azide. Under these conditions, the

TABLE

I

PURIFICATION

OF KALLIKREIN

Steps of purification

-4280

BAEE hydrolysis

Specific activity

&moI/min)

(U/*zsO)

-

Serum Chromatography on QAE-Sephadex Chromatography on Molselect CM-50 Affinity chromatography on SBTI-AHSepharose 4-B

Purification index of esterase actwity

Yqeld of esterase activity

Kininogenase activity (pg bradykinin

(96)

ewiv.l*2so)

4550 685

21.8 17.0

0.0048 0.0262

1 5.5

100 82.1

0.032 -

102

14.5

0.1421

29.6

66.5

0.139

0.269

5.0

18.58

3871

22.9

58.6

326

loo80604020-./';;: 234567

8

9

10

PH

Fig. 3. pHStability of kallikrein. A 0.21111amount of the enzyme was kept for 30 min at 25OC in solutions of various pHs (indicated on the abscissa). Fresh enzyme preparations obtained by affinity chromatography in 0.3 M NaCl/HCl. pH 3.5, were used. The pH of the solution was adjusted to 2.0-3.0 by adding 0.02 M HCl; to 4-8. by adding 0.05 M Tris-HCl buffer. pH 8.0; to 9-10. by adding 0.2 M Tris-HCl buffer, pH 10.3. The total volume of each sample was 0.4 ml. BAEE-esterase activity was measured at PH 8.0.

enzyme retained of the and e&erase for month. Kallikrein was storage for min in M Tris-HCl phosphate pH 8.0, 25°C it lo-15% of initial activity; when the temperature was increased to 37, 55 and it lost 55, 80 95% of initial activity, respectively. The pH-stability curve kallikrein is presented in Fig. It can seen that enzyme was stable at 5-7, whereas more acid more alkaline values its was decreased. The enzyme BAEE TAME with KM of X 1O-4 1.1 X M respectively. Synthetic ester (TLME) hydrolyzed by kallikrein three slower than esters; for TLME found to 3.6 X M. The of our on substrate of toward synthetic are in agreement the data in literature [ 81. kallikrein hydrolyzed in plasma, as as partially purified kininogen also, converted kallikreinogen active kallikrein [ did inhibit the of enzyme. SBTI to be kallikrein inhibitor with Ki of X 10e7 In contrast the data Colman et [8], in experiments the of was inhibited TLCK (Ki 0.17 X M). Comparative were obtained suppressing kallikrein by and other proteinase of Trasylol type (Gordox, Pantripin) which in use; of the tested were to be inhibitors of kallikrein, a Ki 1.8 X M, 2.3 lo-’ M, X lo-* and 1.5 lo-’ M,

1 2 3 4

Colman, R.W.. Mattler, L. and Sherry. S. (1969) J. Clin. Invest. 48, 11-22 McConnel, D.J. and Mason, B. (1970) Br. J. Pharmacol. 38.49e-495 Laufer. A.L., Gulikova. O.M. and Pashkina. T.S. (1969) Biokhimiya 54,3-12 Takahashi. II., Nagasawa. S. and Suzuki. J. (1972) J. Biochem. 71. 471-483

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