Baustert J H, Wolfbeis O S, Moser R, Koller E
Analytical Division, Karl-Franzens University, Graz, Austria.
Anal Biochem. 1988 Jun;171(2):393-7. doi: 10.1016/0003-2697(88)90503-9.
A direct and continuous kinetic method for the fluorometric determination of alpha-chymotrypsin and trypsin is described, and 2-aminoacridone (2-AA) is introduced as a promising new fluorophore in analytical biochemistry. N-Succinyl- and N-glutaryl-phenylalanine as well as N-benzoylarginine were coupled to 2-AA via a peptide bond and the resulting fluorogenic substrates are shown to be cleaved by the two enzymes. Since the substrate and product of hydrolysis have quite different spectral properties, the increase in the long-wave fluorescence of 2-AA (measured at 570 nm under 450-nm excitation) is a parameter for the enzyme activity. Chymotrypsin (0.5 microgram/ml) and trypsin (0.1 microgram/ml) were detectable in a 3-min assay. The major advantages of the new substrates over existing ones are the analytical wavelengths which are distinctly outside the background fluorescence of most biological matter and the somewhat faster reaction rates which can reduce the time of analysis.
描述了一种用于荧光测定α-糜蛋白酶和胰蛋白酶的直接连续动力学方法，并介绍了2-氨基吖啶酮（2-AA）作为分析生物化学中一种有前景的新型荧光团。N-琥珀酰基-和N-戊二酰基-苯丙氨酸以及N-苯甲酰基-精氨酸通过肽键与2-AA偶联，结果表明所得的荧光底物可被这两种酶裂解。由于水解的底物和产物具有截然不同的光谱特性，2-AA长波荧光的增加（在450 nm激发下于570 nm处测量）是酶活性的一个参数。在3分钟的测定中可检测到糜蛋白酶（0.5微克/毫升）和胰蛋白酶（0.1微克/毫升）。与现有底物相比，新底物的主要优点是分析波长明显超出大多数生物物质的背景荧光，以及反应速率稍快，这可以缩短分析时间。
