Document Detail

Use of enhanced green fluorescent protein to determine pepsin at high sensitivity.
MedLine Citation:
PMID:  15840498     Owner:  NLM     Status:  MEDLINE    
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.
Ajamaluddin Malik; Rainer Rudolph; Brigitte Söhling
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Analytical biochemistry     Volume:  340     ISSN:  0003-2697     ISO Abbreviation:  Anal. Biochem.     Publication Date:  2005 May 
Date Detail:
Created Date:  2005-04-20     Completed Date:  2005-09-09     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0370535     Medline TA:  Anal Biochem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  252-8     Citation Subset:  IM    
Institut für Biotechnologie, Martin-Luther Universität Halle, Kurt-Mothes-Str. 3, 06120 Halle, Germany.
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MeSH Terms
Enzyme Activation
Green Fluorescent Proteins / metabolism*
Pepsin A / analysis*
Pepsinogen A / metabolism
Protein Denaturation
Protein Renaturation
Sensitivity and Specificity
Reg. No./Substance:
0/enhanced green fluorescent protein; 147336-22-9/Green Fluorescent Proteins; 9001-10-9/Pepsinogen A; EC A

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