서지주요정보
Studies on the immobilized urokinase = 고정화 유로키나제의 연구
서명 / 저자 Studies on the immobilized urokinase = 고정화 유로키나제의 연구 / Hyun-Pyo Kim.
저자명 Kim, Hyun-Pyo ; 김현표
발행사항 [서울 : 한국과학기술원, 1984].
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4102345

소장위치/청구기호

학술문화관(문화관) 보존서고

DBE 8403

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초록정보

Plasminogen activators are serine proteases with trypsin-like specificity which convert the inactive zymogen plasminogen to enzymatically active plasmin and thereby initiate fibrinolysis $\mbox{\underline{in}}$ $\mbox{\underline{vitro}}$ and $\mbox{\underline{in}}$ $\mbox{\underline{vivo}}$. Among them, human urinary type plasminogen activator, known as urokinase (EC.3.4.99.26) has been used clinically for treatment of thrombosis and/or vascular obstructions. The half-life of urokinase $\mbox{\underline{in}}$ $\mbox{\underline{vivo}}$, however, is very short and hence large amount of urokinase and repeated injections are required to maintain enough fibrinolytic potential $\mbox{\underline{in}}$ $\mbox{\underline{vivo}}$, when urokinase is used for the clinical treatment. In addition, many prosthetic materials such as artificial organs are widely used. Because of development of thrombus on the surfaces of body prosthetic material contact to blood, continuous use in body has severe limitations and application of urokinase to artificial organs is gradually required. To improve these disadvantages and to develope clinical application of urokinase requirements to these fundamental scientific knowledge on urokinase, immobilization/conjugation were attempted in studying 1) model study of immobilization of urokinase on Sepharose matrix, 2) development of conjugated form of urokinase on soluble dextran, 3) development of immobilized urokinase on cellulose hollow fiber membrane used in artificial kidney set. Immobilization of urokinase on insoluble Sepharose gel was succeeded as follows ; To the CNBr-activated Sepharose gel, urokinase was directly coupled. The resulting urokinase showed 37\% caseinolytic activity compared with initial loading amount, and 14.4 CTA units of specific activity. And Sep-$C_n$-COOH-UK was prepared by coupling to NOS-activated Sep-($CH_2$)n_COOH which was previously synthetized from CNBr-activated Sepharose gel and $NH_2$-($CH_2$)n_COOH. The resulting immobilized urokinases having the spacers, $NH_2$-($CH_2$)n_COOH, showed 33-75\% recovery yields of caseinolytic activity and the maximum value, 75\% recovery in the case of Sep-$C_5$-COOH-UK. The specific activities of the Sep-Cn-COOH-UK series were revealed to be 31.6-41.0 CTA units. Finally, to increase the length of the spacers used, $\beta$-alanine was coupled to NOS-activated Sep-($CH_2$)n_COOH and Sep-($CH_2$)n_CONH-$(CH_2)_2$-COOH were obtained. To NOS-activated $\beta$-alanine linked gel, urokinase was immobilized and Sep-Cn_$C_2$- COOH-UK were synthetized which showed 52-68\% caseinolytic activity and 41.4-64.0 CTA units of specific activities. The specific activity of the SepCn_COOH-UK series increased to n=7, and for the Sep-Cn_$C_2$- COOH-UK series, the specific activity increased to n=3 and decreased thereafter. The differences of the length of the spacers used in two serieses to express the highest specific activity may lie in the differences of hydrophilicity of two serieses of spacers and partly in the interaction between matices. The immobilized urokinases to Sep($CH_2$)n_COOH on Sep-($CH_2$)n_CONH-$(CH_2)_2$-COOH showed higher retention yields and much higher specific activities than directly coupled urokinase. It may be due to the steric hinderance released by interposing spacers between enzyme and matrix. It has been attempted to conjugate urokinase to soluble dextran in two different methods, CNBr-activation and oxirane-activation methods. The dextran-urokinase conjugate synthetized by CNBr-activation is appeared to be soluble in aquous solvent and showed 7\% retained fibrinolytic activity and 15.2\% specific activity. And dextran-urokinase conjugate by oxirane-activation had 10-11\% retained fibrinolytic activity and 38\% specific activity, although the conjugation yield as calculated on the basis of protein bound to dextran molecule was lower than that of dextran-urokinase conjugate by CNBr-activation. But above two dextran conjugated urokinases retained almost 100\% of specific activity measured in chromogenic assay method using S-2444. Therefore it is concluded that dextran-urokinase conjugate also experienced severe steric hinderance. But they showed higher stability than the native urokinase. Against inhibition of 6-AHA and plasma, the conjugate was more resistant than the native form, especially inhibition test with $\alpha$2-macrogrobulin portion. It is considered that the differences of the inhibition degree between these two forms of urokinase may be partly due to the steric restrain and partly due to the loss of the inhibitor binding site in the urokinase molecule. And it is revealed that the $\mbox{\underline{in}}$ $\mbox{\underline{vitro}}$ biological half-life time, in human plasma of the conjugate was 20-24 hours, but 30-60 min for the native urokinase. It is seemed that the modified urokinase having good efficiency can be synthetized with conjugation to soluble dextran. In the case of immobilization to cellulose hollow fiber, the expressed fibrinolytic activity of cellulose immobilized fiber increased with the loading amount of urokinase. The resulting urokinase immobilized fiber actually lysed fibrin clot and showed 0.019-0.038 CTA unit/cm fiber of fibrinolytic activity. In contrast to that, the expressed fibrinolytic activity was 0.017-0.030 CTA unit/cm fiber of the simply adsorbed urokinase cellulose fiber, which was appeared to be unstable in storage at 4$^\circ$C. But no decrease of fibrinolytic activity of the urokinase immobilized fiber was observed in storage at 4$^\circ$C. The repeat use test showed higher stability of cellulose immobilized urokinase. And it is observed by scanning electron microscope that platelets are adsorved on the surface of the nontreated fiber, but rare platelets are seen on the urokinase immobilized fiber. Thus, it is suggested that immobilization of urokinase can be used to reduce the thrombogenicity of the surfaces of artificial medical materials.

서지기타정보

서지기타정보
청구기호 {DBE 8403
형태사항 x, 148, [5] p. : 삽도 ; 26 cm
언어 영어
일반주기 Includes appendix
저자명의 한글표기 : 김현표
지도교수의 영문표기 : Si-Myung Byun
지도교수의 한글표기 : 변시명
학위논문 학위논문(박사) - 한국과학기술원 : 생물공학과,
서지주기 Reference : p. 136-148
주제 Proteinase.
고정화 효소. --과학기술용어시소러스
플라스미노겐. --과학기술용어시소러스
프로테이나아제. --과학기술용어시소러스
Immobilized enzymes.
Plasminogen.
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