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. 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.
Malik
Ajamaluddin
A
Institut für Biotechnologie, Martin-Luther Universität Halle, Kurt-Mothes-Str. 3, 06120 Halle, Germany.
Rudolph
Rainer
R
Söhling
Brigitte
B
D000818:Animals; D004789:Enzyme Activation; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D010434:Pepsin A / Q000032:analysis*; D020116:Pepsinogen A / Q000378:metabolism; D011489:Protein Denaturation; D020673:Protein Renaturation; D012680:Sensitivity and Specificity; D013552:Swine
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D020116:Pepsinogen A; D010434:Pepsin A