Radiolabeled biotinyl peptides as useful reagents for the study of proteolytic enzymes.
A radiometric assay for studying the proteolytic activity of endopeptidases using a radiolabeled biotinyl peptide substrate is described. The method relies on the use of a peptidyl substrate incorporating susceptible bonds located between a biotinyl group at one end and a radioiodinated group at the other end. Two tyrosine-containing peptidyl substrates, a fragment of rat plasma kallikrein and a derivative of Leu-enkephalin, were coupled to biotin by reacting with N-hydroxysuccinimidyl-6-(biotinamido) hexanoate. The enzymatic activity is measured by the release into solution of the radiolabeled peptide fragment following selective retrieval, using immobilized avidin, of the biotinyl undigested substrate and the unlabeled biotinyl peptide fragment. This study illustrates that retrieval can be done either before incubation with the enzyme by immobilizing the labeled substrate onto avidin-agarose or, alternatively, after incubation by treating the resulting digest with immobilized avidin. The identity of the labeled released peptides and hence the sites of cleavage can be obtained following separation by RP-HPLC. This assay, in addition to allowing proteolysis to occur with the substrate either in solution or immobilized, is rapid, sensitive (less than 1 pg/ml of trypsin), reproducible, and applicable to the detection of members of all endopeptidase classes. Furthermore, incubation of the radiolabeled biotinyl peptide substrates with enzymes immobilized within polyacrylamide gel slices can be used to detect proteolytic activity following electrophoretic separations under denaturing or nondenaturing conditions.
Basak
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A
J. A. De Séve Laboratory of Molecular Neuroendocrinology, Clinical Research Institute of Montréal, Quebec, Canada.
Boudreault
A
A
Jean
F
F
Chrétien
M
M
Lazure
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007457:Iodine Radioisotopes; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010447:Peptide Hydrolases / Q000032:analysis*; D010455:Peptides / Q000032:analysis* / Q000138:chemical synthesis; D011489:Protein Denaturation; D051381:Rats
D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D010455:Peptides; D001710:Biotin; D010447:Peptide Hydrolases