Urokinase (EC 3. 4. 99. 26), a plasminogen activator, converts plasminogen to plasmin which has the ability to dissolve the fibrin clot. Hence it is generally used for the clinical treatment of the various vascular diseases and for the preparation of the thromboresistant material such as artificial organ and suture material. Through the urokinase is used as the most frequently prescribed agent and also there is an increasing need to develope the better medical material with high antithrombogenicity, the maintenance of the enzyme activity over a long period in vivo is most difficult to attain and the purification of the urokinase causes difficulties.
Several attempts have been tried to improve such materials by using immobilization technique so far. However, the recovery of activity or steric hinderance after the immobilization causes many problems in the urokinase immobilization.
In this study, therefore, the immobilization of the urokinase was carried out by various methods and also the effects of spacer molecule on the accessibility of substrate, a macromolecular plasminogen, to the coupled urokinase to macromolecular matrix were examined by interposing different length of hydrocarbon chain between enzyme and the matrix back-bone as a model study.
The human plasminogen, substrate for urokinase, was purified from Human Cohn Fraction Ⅲ. The Cohn Ⅲ solution was applied to the lys-Sepharose affinity column and the plasminogen was dialyzed, followed by freeze-drying. The final products were used in the assay of the urokinase.
The urokinase was immobilized directly to the CNBr-activated Sepharose 4B as well as to the Sepharose 4B which contained different length of hydrocarbon chain as spacers. For the direct immobilization of urokinase, Sepharose 4B was activated by CNBr, followed by coupling with urokinase at pH 7.5. For indirect coupling two methods were applied. Firstly, different length of aminoalkyl carboxylic acid $(NH_2-(CH_2)_x-COOH)$ was coupled to the CNBr-activated Sepharose 4B, followed by immobilization of urokinase after the COOH groups of aminoalkyl carboxylic acid was activated with N-hydroxy succinimide. Secondly, spacer length of the above alkylated Sepharose 4B, which was prepared in the first indirect method, was increased by reacting β-alanine, and the urokinase was immobilized to the matrix after the COOH of β-alanine was activated with N-hydroxysuccinimide.
Direct attachment of the urokinase to Sepharose 4B showed 35-40% recovery of the urokinase activity, while the urokinase immobilized on the hydrocarbonated Sepharose retained higher original activity as follows :
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In the immobilization process for urokinase, the use of the carbodiimide as condensation agent destroyed most of urokinase activity, while the most of pronase was not lost by the same treatment.
The dramatic effects of urokinase activity in the increasing the extension arm lengh can be obtained by the relief of steric restriction imposed by the matrix and in part by the increased flexibility and mobility of the enzyme as it portrudes further into the solvent. As can be expected, the immobilized urokinase which had $(CH_2)_5$ group in their series and $(CH_2)_{2+2}$ as well as $(CH_2)_{3+2}$ groups as spacer showed the highest specific activity in their series, respectively, on the basis of protein bound which was analyzed by amino acid analyzer. Further extension of spacer length more than $(CH_2)_2$ or $(CH_2)_{3+2}$ in their serieses reduced the enzyme activity, probably due to the folding effect of the spacer.
Among the various immobilized urokinase which were prepared in this study, the direct coupled urokinase and the immobilized urokinase to caprylic acid-coupled Sepharose 4B were characterized for pH dependence, temperature dependence and storage stability with the comparison of the soluble urokinase. The following Table illustrated the results.
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유로키나제(urokinase)는 사람의 오줌으로 부터 얻어지는 효소로써 혈액속의 플라즈미노겐 (plasminogen) 을 Fibrin blood clot 을 용해시키는 작용이 있는 플라즈민 (plasmin)으로 활성화 시킨다. 그러므로 유로키나제는 뇌혈전증, 폐전색증, 심근경색증, 고혈압 등의 임상적인 치료제로 사용되며 또한 근래에 많이 필요성이 거론되고 있는 항혈액응고 물질 (Antithrombogenic material) 을 만드는데 사용 가능성이 높아지고 있다. 불행하게도 생체내에서의 유로키나제의 활성 감소가 아주 커 사용이 제한되고 있으나 이런 활성 감소의 방지와 항혈액응고 물질을 만드는 방법으로 유로키나제의 고정화가 좋은 방법이 될 수 있다. 그러므로 여러가지 방법에 의한 고정화 유로키나제의 제법과 효소와 담체(matrix) 사이에 여러가지 길이의 Spacer를 넣어서 고정화를 하여 담체에 의한 Steric problem 와 프라즈미노겐에 대한 접근력 (accessibility)등을 향상시키고져 담체를 세파로즈 4B (Sepharose 4B) 로 한 모형 연구를 행하였다. 세 가지 다른 방법에 의하여 제조된 고정화 유로키나제는 다음과 같다. 첫째 세파로즈 4B 를 CNBr 로 직접 활성화하여 유로키나제를 고정시키는 방법, 둘째 spacer 로 여러가지 길이의 amino alkyl carboxylic acid 를 갖는 세파로즈 4B 를 N-hydroxysuccinimide 로 활성화 하여 유로키나제를 고정시키는 방법, 세째 amino alkyl carboxylic acid 를 갖는 세파로즈 4B 에, 다시 β-alanine 를 붙여서 spacer 길이를 더 길게한 후 N-hydroxy Succinimide 를 사용하여 유로키나제를 고정화 하는 방법이었다. 담체인 세파로즈 4B 에 직접 고정화하는 방법은 활성 유로키나제의 회수 (recovery) 가 35-40% 이었고 spacer 를 갖고 있는 담체에 고정화 하는 방법은 회수율이 30-80% 이었다. 또한 고정화 유로키나제의 비활성도 (specific activity)는 직접 담체에 고정화한 유로키나제(40 - 58%) 보다 spacer가 유로키나제와 담체사이에 있는 고정화 효소 (90-200%) 가 훨씬 높다. 또한 비활성도가 spacer의 methylene group 의 수 2에서 5까지는 증가하다가 7에서는 급격한 감소를 보였고 β-alanine의 더 증가된 spacer 계열에서는 2+2 와 3+2 가 높고 5+2 에서는 급격한 감소를 보였다. 그러나 이렇게 N-hydroxy Succinimide 을 사용하여 고정화한 유로키나제에 비하여 축합제 (condensation agents) 로 CMC 나 EDC 를 사용한 고정화 유로키나제의 회수율은 0-5%로 아주 낮았다. 또한 이렇게 제조된 두 가지의 고정화 유로키나제 (담체에 직접 고정화된 유로키나제, spacer 로 amino caprylic acid 가 붙은 고정화 유로키아제) 와 유로키나제 (soluble urokinase) 의 최적 pH 는 유로키나제와 세파로즈에 직접 결합된 유로키나제가 7.5 - 8.0 인데 비하여 spacer가 있는 고정화 유로키나제는 7.5 - 9.0 의 넓은 최적 pH 를 나타내었다.
최적 온도는 세 가지가 전부 35-40℃였고 체액과 등장인 식염용액하에서의 안정도는 담체에 직접 결합된 유로키나제에 비해 spacer 가 있는 고정화 유로키나제가 훨씬 높았고 spacer 가 있는 고정화 유로키나제가 열 (thermodenaturation)에도 아주 강하였다.
