Straight D L, McKee P A
Biochemistry. 1982 Sep 14;21(19):4550-6. doi: 10.1021/bi00262a006.
We have evaluated intrinsic protein fluorescence as a method for investigating the reactions of alpha 2-macroglobulin (alpha 2M) with proteases and amines. Changes in fluorescence intensity of alpha 2M in the presence of proteases and amines were shown to correlate with structural and functional changes in the alpha 2M molecule. By intrinsic fluorescence we found that 2 mol of trypsin bound to 1 mol of alpha 2M whereas thrombin and plasmin each bound in a stoichiometry closer to 1:1. Studies showed that changes in fluorescence caused by ammonium ion paralleled the loss of the ability of alpha 2M to protect trypsin from soybean trypsin inhibitor. The exposure of sulfhydryl groups on alpha 2M by a small organic amine (methylamine) also correlated with fluorescence change that could be quantitatively eliminated by prior reaction of alpha 2M with trypsin. Cleavage of alpha 2M by four serine proteases (plasmin, thrombin, trypsin, and elastase) as determined by sodium dodecyl sulfate gel electrophoretic analyses and the binding of plasmin and thrombin as measured by macromolecular inhibitor assays corresponded to the increase in fluorescence intensity. In addition, the rate of thrombin inhibition for clotting fibrinogen was the same as the rate of fluorescence change observed when thrombin was incubated with alpha 2M. Our results indicate that intrinsic protein fluorescence is an easy and rapid technique for assessing both qualitative and quantitative aspects of protease-alpha 2M interactions.
我们已评估了蛋白质固有荧光，将其作为一种研究α2-巨球蛋白（α2M）与蛋白酶及胺类反应的方法。结果表明，在蛋白酶和胺类存在的情况下，α2M荧光强度的变化与α2M分子的结构和功能变化相关。通过固有荧光我们发现，2摩尔胰蛋白酶与1摩尔α2M结合，而凝血酶和纤溶酶的结合化学计量比均更接近1:1。研究表明，铵离子引起的荧光变化与α2M保护胰蛋白酶免受大豆胰蛋白酶抑制剂抑制的能力丧失情况相似。一种小有机胺（甲胺）使α2M上的巯基暴露，这也与荧光变化相关，而该荧光变化可通过α2M预先与胰蛋白酶反应而被定量消除。通过十二烷基硫酸钠凝胶电泳分析测定的四种丝氨酸蛋白酶（纤溶酶、凝血酶、胰蛋白酶和弹性蛋白酶）对α2M的切割，以及通过大分子抑制剂测定法测量的纤溶酶和凝血酶的结合，均与荧光强度的增加相对应。此外，凝血酶抑制纤维蛋白原凝固的速率与凝血酶与α2M孵育时观察到的荧光变化速率相同。我们的结果表明，蛋白质固有荧光是一种用于评估蛋白酶与α2M相互作用的定性和定量方面的简便快速技术。
