In this study, the large-form urokinase (L-UK), which had been demonstrated to be more effective in treating thrombosis, was isolated from a crude preparation of local source to electrophoretical and
immunological homogeneity simply by gel filtration and affinity
chromatography.
The circular dichrois spectrum indicated that an essentially random coil
and β-conformation are involved in the secondary structure of this
enzyme. This result is in good agreement with the typical properties of
urokinase observed, namely stable in higher temperature, and in the
presence of 6 M urea or 0.1% sodium dodecyl sulfate, but loss in activity
at a very low salt concentration.
A simple fluorometric method for microassay of sugar content (including
neutral and amino sugars)in glycoprotein (e.g. urokinase or ovalbumin)(was
developed. By this method, less than 100 pmol of sugar contained in
glycoprotein could be detected. On the orther hand, an established
double-isotope labeling technique was modified to meet the requirement for
micro-assay of amino acid content of protein.
When the purified L-UK was further separated by preparative
isoelectric-focusing electrophoresis, three isozymes different in pI
values (9.9, 8.9 and 8.35) could be obtained. The amino acid compositions
and tryptic peptide maps of L-UK and separated L-UK isozymes were assessed
and compared with those ofsmall-form urokinase (S-UK) from Mochida
(Japan). It was found that there were great similarties among these enzyme
proteins and the homology of L-UK and S_UK in primary structure was
proposed.
The sugar content as well as some other molecular and kinetic parameters
(dusing benzoyl_L-valyl-glycyl-L-argininp-nitroanilide (BVGANA) or
plasminogen as the substrate) of L-UK and L-UK isozymes are compared in
the table on next page.
From these data, several valuable findings as listed in the following were
obtained.
1)Although the total number of acidic amino acid residues was 1.1 to 1.2
fold larger than the sum of basic aminoacid residues, the high pI values
of L-UK isozymes (8.4 to 9.9) suggested the presence of acidic amino acid
in their amide form in a large proportion.
2)The difference in pIs of L-UK isozymes was mainly due to the difference
in sialic acid content.
3)Glucose (or sucrose)was found in L-UK siozymes, but not in L-UK itself.
It indicated that sugar might be adsorbed onto the enzyme molecule in a
low ionic strength and high sugar content medium.
4) A sugar content pattern (but not amino acid content) distinctly
different from those published by others was obtained. This discrepancy
might be due to the difference in the sources of enzyme preparations.
5) The similarity in the kinetic parameters suggeted that L-UK and L-UK
isozymes have a similar affinity toward the substrate used (BVGANA and
plasminogen).
In the isolation of L-UK, a buffer solution carrying higher ionic strengh
(such as 0.1 M phosphate buffer containing 0.1 M or higher NaCl) must be
used for eluting the enzyme from a Sephadex gel column. Otherwise, the
L-UK may dissociate or degrade to forms of lower molecular weight (e.g.
S-UK ) with simultaneous polymerization. This is to our opinion, one of
the important reasons why variable forms of UK in various ratioes have
been reported previously.
In the assay of urokinase activity, the conventional fibrin plate method
was modified to be a more reproducible and convenient procedure by
punching holes for sample application on a agar-containing fibrin plate.
In addition, a more specific enzyme immunoassay method was developed. The
anti-L-UK was isolated from the rabbit antiserum and coupled with
horseradish peroxidase as the marker enzyme. The enzyme conjugate thus
preoxidase as the marker enzyme. The enzyme conjugate thus prepared was
used for the quantitative analysis of urokinase by a competitive
immunoassay in the presence of immobilized UK (UK-Sepharose). This method
was comparable in sensitivity to the traditional fibrinolytic assay and an
established radio-immunoassay.
尿激□(urokinase)乃臨床上重要之血管栓塞治療劑，目前本省年產此□之粗製 品約
值新台幣兩億元。本論文之研究主旨在於分離出較具醫療效果的重型尿激□ (large
form urokinase)進而深入地探討此型尿激□之分子與酵素特性及產生輕型尿 激□
(small form urokinase)之可能原因，俾便對省產此一酵素有更明確的認識。最 後敘
述一種新發展而具實用性的尿激□酵素免疫分析法(enzyme immunoassay)。
省產粗製尿激□（比活性約為10,000國際單位╱毫克蛋白質）可簡單的經膠體過 濾層
析及親和性層析法而分出重型尿激□，比活性約為90,000國際單位╱毫克蛋白質 ，其
純度經膠體電泳法及免疫分析證明為均質物。
由圓光二色性（Circular dichroism）光譜分析顯示重型尿激□之二級構造以雜 亂捲
曲(random coil)及β－構形(β-conformation)為主，這種特徵與尿激□已知的 一些
特性頗為吻合。例如，在高溫中較一般酵素安定，於含有六莫耳尿素(6 M urea) 或十
分之一重量百分濃度的十二烷基硫酸鈉(0.1% sodium dodecyl sulfate (SDS))溶液
中亦能保持活性，但當溶液之鹽度過低時其活性會明顯的降低。
為瞭解尿激□（屬於醣蛋白）的化學組成，發展出一種簡單而適用於醣蛋白中醣 質定
性與定量之螢光分析法。根據此法，可檢出醣蛋白中醣質定性與定量之螢光分析 法。
根據此法，可檢出醣蛋白中低至1×10-10莫耳濃度醣質的存在，而若含量在1× 10-9
莫耳以上時即可精確加以定量。另外，本文亦述及一種經改良而適合於醣蛋白質 氨基
酸組成微量分析的雙同位素標幟法(doubled-isotope labeling mehtod)。
純重型尿激□可進一步用等電焦集電泳法(isoelectric ocusing electrophoresis)
分離出三種同功□(isozymes)，等電點分別為9.9（約佔70%）8.9及8.35。然而 此等
異構□及重型尿激□間之氨基酸組成及胜□圖(peptide map)頗為類似；進而與 得自
日本持田公司(Mochida Co.)之輕型尿激□比較亦極相近，因而推測重、輕型間 之一
級構造具有相當程度的對應性，對於輕型尿激□係來自重型尿激□之理論提供一 重要
佐證。
其他有關重型尿激□及其異構□的醣質組成及其分子和動力學參數整理並比較於 下表
：
綜合上述數據，有幾項主要發現如下：
雖然酸性氨基酸總含量為鹼性氨基酸總含量約1.1至1.2倍，但三種異構□之等電 點
卻偏高(8.35∼9.9)，顯示分子內有相當程度的酸性氨基酸以醯胺化的型式存在 。
異構□間等電點之差異主要在於涎酸(sialic acid)含量之不同。
葡萄糖存在於異構□，而不存在於原來的重型□本身，顯示在低離子強度及高 醣含
量的電泳介質中，醣質可能與酵素分子結合。
尿激之醣質分析結果與已有的數據並不一致（尤其是半乳糖的含量），這種差異
可能是酵素來源不同所引起，不過氨基酸組成並無此現象。
重型尿激□與其異構□間的動力學參數(kinetic parameters)頗為類似，顯示 彼等
對於基質之親和力大致相同。
欲單離重型尿激□，於膠體過濾法(gel filtration)操作中應使用離子強度較高 的緩
衝液（例如十分之一莫耳磷酸緩衝溶液含十分之一莫耳或更高的氯化鈉）。否則 ，重
型尿激□發生解離或分解 (degrade)而出現分子量較低的衍生物（類似於輕型尿 激□
）而且聚合亦會同時發生。此種現象可以說明為何前人的報告認為尿激□有多種 型式
以不同比例出現。
在尿激□的活性分析方面，本文中將傳統的纖維蛋白平板法(fibrin plate method)
加以改良，首先添加洋菜(agar)於纖維蛋白平板中令其固化，然後加以打洞，再 將酵
素樣品注入其中，由於酵素之擴散與作用相當均勻因而可縮短反應時間並提高分 析上
之再現性。另外又發展一種特異性高的酵素免疫分析，此法係將過氧化□
(peroxidase)標幟在尿激□濃度之目的，其靈敏度雖與傳統的纖維蛋白分析或已 有之
放射性免疫分析法(radio-immunoassay)近似，但較一般的酯解(esterolytic)或 醯胺
解(amidolytic)分析法為高。