Malik Ajamaluddin, Rudolph Rainer, Söhling Brigitte
Institut für Biotechnologie, Martin-Luther Universität Halle, Kurt-Mothes-Str. 3, 06120 Halle, Germany.
Anal Biochem. 2005 May 15;340(2):252-8. doi: 10.1016/j.ab.2005.02.022.
A fluorometric assay for pepsin and pepsinogen was developed using enhanced green fluorescent protein (EGFP) as a substrate. Acid denaturation of EGFP resulted in a complete loss of fluorescence that was completely reversible on neutralization. In the proteolytic assay procedure, acid-denatured EGFP was digested by pepsin or activated pepsinogen. After neutralization, the remaining amount of undigested EGFP refolded and was determined by fluorescence. Under standard digestion conditions, 4.8-24.0 ng pepsin or pepsinogen was used. Using porcine pepsin as a standard, 38+/-6.7 ng EGFP was digested per min-1 ng pepsin-1. Activated porcine pepsinogen revealed a similar digestion rate (37.2+/-5.2 ng EGFP min-1 ng activated pepsinogen-1). The sensitivity of the proteolysis assay depended on the time of digestion and the temperature. Increasing temperature and incubation time allowed quantification of pepsin or pepsinogen in a sample even in the picogram range. The pepsin-catalyzed EGFP digestion showed typical Michaelis-Menten kinetics. Km and Vmax values were determined for the pepsin and activated pepsinogen. Digestion of EGFP by pepsin revealed distinct cleavage sites, as analyzed by SDS-PAGE.
开发了一种以增强型绿色荧光蛋白（EGFP）为底物的胃蛋白酶和胃蛋白酶原荧光测定法。EGFP的酸变性导致荧光完全丧失，而中和后荧光可完全恢复。在蛋白水解测定过程中，酸变性的EGFP被胃蛋白酶或活化的胃蛋白酶原消化。中和后，剩余未消化的EGFP重新折叠并通过荧光测定。在标准消化条件下，使用4.8 - 24.0 ng胃蛋白酶或胃蛋白酶原。以猪胃蛋白酶为标准，每分钟每1 ng胃蛋白酶可消化38±6.7 ng EGFP。活化的猪胃蛋白酶原显示出相似的消化速率（每分钟每1 ng活化的胃蛋白酶原可消化37.2±5.2 ng EGFP）。蛋白水解测定的灵敏度取决于消化时间和温度。提高温度和延长孵育时间能够对样品中的胃蛋白酶或胃蛋白酶原进行定量，甚至可达到皮克级别。胃蛋白酶催化的EGFP消化呈现典型的米氏动力学。测定了胃蛋白酶和活化胃蛋白酶原的Km值和Vmax值。通过SDS - PAGE分析，胃蛋白酶对EGFP的消化显示出不同的切割位点。
