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葡萄球菌激酶活性的定性和定量测定(作者译)

[Qualitative and quantitative determinations of staphylokinase-activity (author's transl)].

作者信息

Papke G, Blobel H

出版信息

Zentralbl Bakteriol Orig A. 1978 Dec;242(4):456-61.

PMID:219637
Abstract

Staphylokinase (SAK)-activated plasminogen reacted specifically with nitroanilide (N-benzoyl-DL-lysine-4-nitroanilide) to form intensively yellow 4-nitroaniline. This reaction did not occur with staphylococcal proteases. For qualitative SAK-determinations nitroanilide was incorporated in an agar medium. SAK diffused into the medium and caused a distinct change in color from white to yellow. For quantitative SAK-determination the conversion of colorless nitroanilide to yellow 4-nitroaniline was recorded photometrically at 405 nm. The optical density correlated well with SAK-activity of preparations with different degrees of purity (fig. 1, 2). Another quantitative procedure for SAK-activity could be conducted with fibrin in microtiter-plates (Fib-MTT). In this method, after 2-fold dilutions of SAK-preparations fibrinogen (stained blue with astrazonblue) and subsequently thrombin were added. SAK-activity was indicated by lysis of the blue-colored fibrin clots in the microtiter-plates (fig. 3). The Fib-MTT was particularly suitable for measuring wide ranges of SAK-activities.

摘要

葡萄球菌激酶(SAK)激活的纤溶酶原与硝基苯胺(N-苯甲酰-DL-赖氨酸-4-硝基苯胺)发生特异性反应,生成颜色强烈发黄的4-硝基苯胺。葡萄球菌蛋白酶不会发生这种反应。为了定性测定SAK,将硝基苯胺加入琼脂培养基中。SAK扩散到培养基中,导致颜色从白色明显变为黄色。为了定量测定SAK,在405nm处通过光度法记录无色硝基苯胺向黄色4-硝基苯胺的转化。光密度与不同纯度制剂的SAK活性相关性良好(图1、2)。SAK活性的另一种定量方法可以在微量滴定板中用纤维蛋白进行(Fib-MTT)。在该方法中,将SAK制剂进行2倍稀释后,加入纤维蛋白原(用偶氮蓝染色呈蓝色),随后加入凝血酶。微量滴定板中蓝色纤维蛋白凝块的溶解表明了SAK活性(图3)。Fib-MTT特别适用于测量广泛范围的SAK活性。

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