Use of enhanced green fluorescent protein to determine pepsin at high sensitivity

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. K m and V max values were determined for the pepsin and activated pepsinogen. Digestion of EGFP by pepsin revealed distinct cleavage sites, as analyzed by SDS–PAGE.