Inactivation of alpha1-proteinase inhibitor as a broad screen for detecting proteolytic activities in unknown samples.
The need for a quick, simple screening method for the detection of general proteolytic activity prompted us to determine whether cleavage within the reactive site loop region (RSL) of alpha1-proteinase inhibitor (alpha1-PI), a well-characterized member of the serpin family known to be susceptible to proteolytic inactivation, can be utilized for this purpose. Inactivation of alpha1-PI in the RSL region can be measured by loss of residual inhibitory capacity of alpha1-PI against its target proteinase. While we originally utilized this assay to detect a new proteinase from culture supernatants of Porphyromonas gingivalis, the feasibility of extending this assay to scan for proteolytic activity from other systems was also assessed. As an example, we found that the serine proteinase from Staphylococcus aureus (SSP) had virtually the same catalytic efficiency in inactivating alpha1-PI in our assay as it did in the hydrolysis of the synthetic substrate Z-Phe-Leu-Glu-pNA (kcat/Km value of 2 x 10(4) M-1 s-1 vs 2.6 x 10(4) M-1 s-1, respectively). Additionally, in both assays activity could be readily detected in less than a 1 h incubation at SSP concentrations in the picomolar range. This assay is unique in that proteinases which hydrolyze peptide bonds within the RSL of alpha1-PI can readily be detected as measured by loss of alpha1-PI inhibitory activity.
Nelson
D
D
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
Potempa
J
J
Travis
J
J
D000595:Amino Acid Sequence; D002918:Chymotrypsin / Q000032:analysis; D010450:Endopeptidases / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000201:enzymology; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010196:Pancreatic Elastase / Q000032:analysis; D016966:Porphyromonas gingivalis / Q000201:enzymology*; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000379:methods; D013379:Substrate Specificity; D014357:Trypsin / Q000032:analysis; D000515:alpha 1-Antitrypsin / Q000378:metabolism* / Q000494:pharmacology
D009842:Oligopeptides; D000515:alpha 1-Antitrypsin; D010450:Endopeptidases; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D014357:Trypsin