Gel filtration and disc electrophoresis were used as simple and fast techniques for the investigation of the interaction and stoichiometry between UTI and trypsin. UTI appears to possess only a single trypsin binding site. The nature of the interaction between the inhibitor and enzyme appears to be dependent on the concentration ratio of the reactants. When UTI is in excess molar concentration, a single binary complex with trypsin of mol. wt. 95,000 is observed. In the presence of a molar excess of enzyme, this macromolecule is no longer observed, but proteins of mol. wt. 41,000 and 20,000 result. The possibility that UTI may be hydrolyzed to a partially degraded active fragment by the excess enzyme resulting in the formation of a modified inhibitor enzyme complex is proposed

Proksch, G. J.

Routh, J. I.

English

University of Northern Iowa

Proceedings of the Iowa Academy of ScienceVolume 80 | Number Article 51973The Interaction Between the Urinary TrypsinInhibitor and TrypsinG. J. ProkschIndiana University Medical CenterJ. I. RouthUniversity of IowaCopyright © Copyright 1973 by the Iowa Academy of Science, Inc.Follow this and additional works at: http://scholarworks.uni.edu/piasThis Research is brought to you for free and open access by UNI ScholarWorks. It has been accepted for inclusion in Proceedings of the Iowa Academyof Science by an authorized editor of UNI ScholarWorks. For more information, please contact scholarworks@uni.edu.Recommended CitationProksch, G. J. and Routh, J. I. (1973) "The Interaction Between the Urinary Trypsin Inhibitor and Trypsin," Proceedings of the IowaAcademy of Science: Vol. 80: No. 2 , Article 5.Available at: http://scholarworks.uni.edu/pias/vol80/iss2/5.52 The Interaction Between the Urinary Trypsin Inhibitor and Trypsin G. J. PROKSCH1 and J. I. ROUTH2 PROKSCH, G. J. and J. I. ROUTH. The Interaction Between the Urinary Trypsin Inhibitor and Trypsin. Proc. Iowa Acad. Sci. 80( 2): 52-55, 1973. SYNOPSIS: Gel filtration and disc electrophoresis were used as simple and fast techniques for the investigation of the interaction and stoichiometry between UTI and trypsin. UTI appears to possess only a single trypsin binding site. The nature of the interaction between the inhibitor and enzyme appears to be de-The urinary trypsin inhibitor (UTI) is a protein normally present in human urine which inactivates trypsin and which has been reported to possess anticoagulant and antifibrinoly-tic activity,l,2 UTI, which has recently been obtained as an apparently homogeneous protein, is a glycoprotein, contain-ing 10% carbohydrate, and has an estimated mol. wt. of 70,-000.3 The interaction between UTI and trypsin was investi-gated by gel filtration and disc electrophoresis to study the nature of trypsin inactivation by UTI and to serve as a moJel for the inactivation of other enzymes. MATERIALS AND METHODS UTI was prepared from pooled urine from non-proteinuric females in the third trimester of pregnancy according to the method of Proksch and Routh. 3 Twice recrystallized bovine trypsin ( TRL-7LA) and trypsin which had been inactivated by treatment with diisopropylfluorophosphate (DIP-trypsin; TDIP-9GA) were purchased from the Worthington Bio-chemical Corp., Freehold, New Jersey. The nonenzymatic proteins used to calibrate the Sephadex G-100 column were purchased as a kit ( 8190A) from Mann Research Labora-tories, Inc., New York, New York. TRYPSIN AND TRYPSIN INHIBITOR ASSAY Trypsin was assayed by the method of Roth in which the enzymatic hydrolysis rate of N1 cx:-benzoyl-DL-arginine-{1-naphthylamide hydrochloride ( BANA) ( Calbiochem, Los An-geles, California) is measured fluorometrically.4 The trypsin inhibitor content of a given fraction was determined from its ability to reduce the hydrolytic activity of trypsin. The inhi-bition of trypsin by the purified UTI preparation was linear with increasing concentration of the inhibitor between 0 and 95% inhibition. The curve was non-linear between 95% and 100% inhibition. Trypsin concentration was determined using the optical factor of 0.67 mg A-1 at 280 nm.5 1 Department of Clinical Pathology, Indiana University Medical Center, Indianapolis, Indiana 46202. 2 Department of Biochemistry, The University of Iowa, Iowa City, Iowa 52242. pendent on the concentration ratio of the reactants. When UTI is in excess molar concentration, a single binary complex with trypsin of mol. wt. 95,000 is observed. In the presence of a molar excess of enzyme, this macromolecule is no longer observed, but proteins of mo!. wt. 41,000 and 20,000 result. The possibility that UTI may be hydrolyzed to a partially degraded active fragment by the excess enzyme resulting in the formation of a modified inhibitor enzyme complex is proposed. COLUMN CHROMATOGRAPHY The interaction between UTI and trypsin was investigated by gel filtration chromatography according to the method of Andrews.6 A 2.5 x 100 cm column was packed with Sepha-dex G-100 ( 40-120 micron diameter, Pharmacia Fine Chem-icals, Piscatway, N. J.) to a bed height of 85 cm. The col-umn was eluted with a flow rate of 5 ml hrl cm-2 by the reverse flow technique with a constant hydrostatic pressure of 16 cm. The proteins used as standards to calibrate the column were horse apo-ferritin, human gamma globulin, bo-vine serum albumin, ovalbumin, chymotrypsin, cytochrome c (Mann Research Lab) and soybean trypsin inhibitor (Worth-ington Biochemical Corp.) ELECTROPHORESIS Disc electrophoresis was performed using the simplified technique of Clark. 7 The protein bands were identified by fixation and staining with Coomassie brilliant blue dye in 12.5% trichloroacetic acid.8 RESULTS The naturally occurring protease inhibitors may vary with respect to their number of enzyme binding sites. The lower molecular weight inhibitors, such as the soybean trypsin in-hibitor ( SBTI) and Kunitz' s pancreatic inhibitor, are mono-valent,9,10 Other inhibitors with a higher molecular weight, such as duck-egg ovomucoid, may simultaneously bind two moles of trypsin.II The stoichiometry of the interaction be-tween UTI and trypsin was investigated by gel filtration chromatography on a Sephadex G-100 column, calibrated with respect to molecular weight. As shown in Fig. la, UTI was eluted mainly in a single fraction with a peak of 196 ml. This corresponds to the elu-tion of a protein of mol. wt. 70,000 (Fig. 2). When trypsin was added to UTI in either equal or reduced molar concen-trations an additional peak appeared (Fig. 1 b). This frac-1Proksch and Routh: The Interaction Between the Urinary Trypsin Inhibitor and TrypsinPublished by UNI ScholarWorks, 1973INTERACTION BETWEE'.'I UTI AND TRYPSIN 53 l.000 . 800 I ~ .600 ... .. ... z ~ g .400 "' • <C .200 ,, /\ I ~ I \ .. I ' I I .. . . ; \ i : : ! ' ' ' I ' ' ' . I I ' . ! \ ; ~ i \ o I ' I I I f \ l I I \ / \ . ' . ' a ,, ........... ,'' \ .......... .. O--J-~"'T-~-r~-r~-=r==::::.:,:.:;::.:..:...,.._~...,.::::::::: f a .. ... 40 ,400 . 300 ! .200 I .100 40 50 50 60 70 80 90 100 110 120 fllACTION b 60 70 80 90 100 110 120 FRACTION Figure 1. The Elution of UTI and UTI Trypsin Complexes from Sephadex G-100. Fig. la. The dotted line represents the elution of 13 mg of UTI. The solid line represents the elution of 4.1 mg of DIP-trypsin. Fig. lb. The solid line represents the elution of nearly equal molar amounts of UTI and trypsin. UTI ( 3.0 mg) was dissolved in 0.5 ml of buffer ( 0.05 M Tris-HCl, 0.1 M KCl, pH 7.5), and 1.0 mg of trypsin was dissolved in another 0.5 ml of buffer. The two solutions were mixed and allowed to incubate for 10 min. The sample was then applied to the column. The dotted line represents the elution of 2.0 mg of UTI and 1.0 mg of trypsin (an approximate 1.5 molar excess of enzyme) prepared as previously described. The fraction volumes on the abscissa were 3 ml. tion, with an elution maximum at 179 ml, possessed an ap-parent mol. wt. of 95,000 (Fig. 2). Since trypsin has a mol. wt. of 24,000 and UTI has an approximate mol. wt. of 70,-000, a protein of 95,000 mol. wt. would correspond to the elution of a binary complex. These fractions did not possess any additional inhibitor capacity, indicating, therefore, that UTI possesses only a single binding site for trypsin. \~~ ~ 100,000 9' 00'\ EXCESS 3: •••-•••·-·· NATIVE UTI +TRYPSIN ~ •-Z.0~----.s~ NATIVE UTI '.j "UMIN § eo,ooo 41 000 ~. OVALBUMIN ~ ·--·---•••••••••••••·~·~ UTI +EXCESS TRYPSIN ""· CHYMOTRYPSINOGEN A '•~R~PSIN INHIBITOll • CYTOCHROME g 160 200 240 280 ELUTION VOLUME (ml) .,,, Figure 2. Calibration Curve of the Sephadex G-100 Column for the Estimation of the Molecular Weights of the UTI Trypsin Com-plexes . When UTI was incubated with a molar excess of trypsin for 10 minutes before application to the column, a different pat-tern was observed (Fig. lb). No protein was eluted in either the 70,000 or 95,000 mol. wt. zones; instead, two new peaks appeared. The first protein was eluted in a symmetrical peak at 233 ml, which corresponded to a molecule of 41,000 mol. wt. (Fig. 2) . This region did not possess either trypsin or inhibitor activity. The second fraction was eluted in a broad non-symmetrical zone at 295 ml, corresponding to a protein of approximately 20,000 mol. wt. As shown in Fig. la, in-active DIP-trypsin was eluted in this region. A slight amount of trypsin activity was observed in this area. These fractions probably represent the elution of the excess enzyme and any hydrolytic products. The complexes between UTI and trypsin were further studied by disc electrophoresis. At the pH of the running gel buffer, pH 8.1, UTI migrated toward the anode and trypsin moved toward the cathode. A complex between the two pro-teins would thus be expected to possess an intermediate elec-trophoretic mobility. The electrophoresis of UTI with various amounts of trypsin is presented in Fig. 3. UTI (Fig. 3a) ap-pears to contain only a single band of protein. The addition of approximately one-half an equivalent molar amount of trypsin (Fig. 3b) resulted in the appearance of another pro-tein band which was less anionic than UTI. This new protein band probably represents the 95,000 mol. wt. fraction. The addition of a molar excess of trypsin (Fig. 3c) resulted in the complete disappearance of the UTI band, indicating that the UTI preparation contains only inhibitory protein. There was no indication of the 95,000 mol. wt. macromolecule. In-stead, only a broad diffuse band of reduced electrophoretic mobility proteins may be observed near the top of the gel. The disc electrophoresis study confirms the previous gel fil-tration experiments in that the interaction between UTI and 2Proceedings of the Iowa Academy of Science, Vol. 80 [1973], No. 2, Art. 5http://scholarworks.uni.edu/pias/vol80/iss2/554 PRoc. low A AcAD. Ser. 80 ( 1973) Figure 3. Disc Electrophoresis of UTI and UTI Trypsin Com-plexes. a. 50 µg UTI; b. 45 µg UTI, 9 µg trypsin (approximately lf equivalent molar amount of enzyme); c. 50 µg UTI, 40 µg trypsin (approximately 2.5 equivalent molar amount of enzyme); d. 60 µg trypsin. UTI and trypsin were separately dissolved in the Roth trypsin assay buffer, pH 7.8. The solutions were then mixed and allowed to incubate for 5 min. An equal amount of 10% sucrose was then added and the samples were separated electrophoretically for 45 min. with a constant voltage of 20 volts cm-1. trypsin apparently varies with the relative enzyme concen-tration. DISCUSSION When trypsin, mol. wt. 24,000, is ad:led to equal or greater molar amounts of UTI, mol. wt. 70,000, a complex of mol. wt. 95,000 is observed. This new macromolecule does not possess either trypsin or inhibitor activity. UTI apparently has only a single trypsin binding site. . After UTI was incubated with a molar excess concentrat10n of trypsin, different results were obtained. UTI completely disappeared and there was no subsequent appearance of the 95,000 mol. wt. complex. Instead, two different protein frac-tions were observed. One fraction was present as a broad non-symmetrical peak of approximate mol. wt. 20,000, and ap-peared in part to be due to the elution of excess enzyme. The other band which was symmetrical corresponded to the elu-tion of a macromolecule of 41,000 mol. wt. which did not contain any trypsin or inhibitor activity. The addition of in-creasing amounts of UTI to a molar excess of trypsin re-sults in a linear stiochiometric decrease in proteolytic activity between 0 and 95% inhibition. UTI, therefore, is active in the presence of excess enzyme. The only macromolecule lar-ger than trypsin which was observed after incubating UTI with a molar excess of enzyme was the fraction of mol. wt. 41,000. The nature of the interaction between UTI and tryp-sin appears to be dependent on the concentration ratio of the two reactants . The data suggest that UTI may be par-tially digested by the excess trypsin and that this is followed by the subsequent formation of a complex between the par-tially hydrolyzed inhibitor and enzyme. Since trypsin has a mol. wt. of 24,000, such a modified inhibitor would possess an approximate mol. wt. of 17,000. The modification of protease inhibitors by trypsin has been previously observed. Laskowski and Ozawa have reported that soybean trypsin inhibitor may be proteolytically cleaved by trypsin to an active modified form.12 This reaction, how-ever, occurred only under acid conditions, and required only catalytic amounts of enzyme. The proposed hydrolysis of UTI in the present experiment, however, occurred under alkaline conditions and required a molar excess of trypsin. The sig-nificance of the proposed property of UTI to undergo ex-tensive proteolysis and retain activity is at present uncertain. Such a system, however, could represent part of a mechanism for the regulation of antiprotease activity in which the in-hibitor activity may be moderated by proteolytic reactions influenced by relative enzyme concentrations. Such a possi-bility will be explored in future investigations. LITERATURE CITED 1. SCHULMAN, N. R. , and J. Z. HEARON. 1963. Kinetics of con-version of prothrombin to thrombin by biological activators. ]. Biol. Chem. 238:155-164. 2. EGEBLAD, K. , and T. ASTRUP. 1966. Fibrinolysis and the tryp-sin inhibitor in human urine. Scand. ]. Clin . Lab. Invest. 18: 181-190. 3. PROKSCH, G. J., and J. I. ROUTH. 1972. The purification of the trypsin inhibitor from human pregnancy urine. ]. Lab. and Clin. Med. 79:491-499. 4. Rorn, M. 1963. Dosage fluorimetric de la trypsine. Clin. Chim. Acta 8:574-578. 5. Wu, F. C., and M. LASKOWSKI. 1955. Action of the naturally occurring trypsin inhibitors against chymotrypsins ex: and fJ. ]. Biol. Chem. 213:609-619. 6. ANDREWS, P. 1964. Estimation of the molecular weight of pro-teins by Sephadex gel-filtration. ]. Biochem. 91 :222. 7. CLARK, J. T. 1964. Simplified "disc" ( polyacrylamide gel) electrophoresis. Annal. N.Y. Acad. of Science 121:428. 8. CHRAMBACH, A., R. A. REISFELD, N. WYCKOFF, and J. ZAcCARI. 1967. A procedure for rapid and sensitive staining of protein fractionated by polyacrylamide gel electrophoresis. Anal. Bio-chem. 20: 150. 3Proksch and Routh: The Interaction Between the Urinary Trypsin Inhibitor and TrypsinPublished by UNI ScholarWorks, 1973INTERACTION BETWEE'.'! UTI AND TRYPSIN 55 9. KuxITz, ~L and J. H. NORTHRUP. 1936. Isolation from beef pancreas of crystailine trypsinogen. trypsin, a trypsin inhibitor, and an inhibitor-trypsin compound. ]. Gen. Physiol. 19:991-1007. 10. KuKITZ, \L 1946. Crystalline soybean trypsin inhibitor. ]. Gen. Physiol. 29: 149-154. 11. RHODES, M. B., N. BEX;\;ETT, and R. E. FEE;-.;EY. 1960. The trypsin and chymotrypsin inhibitors from avian egg whites. ]. Biol. Chem. 235: 1686-1693. 12. OzAwA, K., and M. LASKOWSKI, JR. 1966. The reactive site of trypsin inhibitors. ]. Biol. Chem. 241:3955-3961. 13. Pn0Ksc11, G. J., J. I. RouTH, J. S. SMITH, and B. S. NEWMAN. 1972. Immunological relationship of the urinary trypsin inhibitor to a pbsma cx:-globulin. Clin Biochem. 5:242-245. 4Proceedings of the Iowa Academy of Science, Vol. 80 [1973], No. 2, Art. 5http://scholarworks.uni.edu/pias/vol80/iss2/5

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