An electroblotting, two-step procedure for the detection of proteinases and the study of proteinase/inhibitor complexes in gelatin-containing polyacrylamide gels

A two-step gelatin/polyacrylamide gel electrophoresis (gelatin/PAGE) proced ure was devised for the detection of proteinases and the study of proteinas e/inhibitor interactions in complex biological extracts. The proteins are f irst resolved by sodium doclecyl sulfate (SDS)-PAGE under reducing or nonre ducing conditions, and electrotransferred into a 0.75 mm-thick accompanying polyacrylamide slab gel containing 0.1% w/v porcine gelatin. The active pr oteinase bands are developed by a gelatin proteolysis step in the accompany ing gel in the presence or absence of diagnostic proteinase inhibitors, all owing the assessment of proteinase classes and the visual discrimination of inhibitor-'sensitive' and -'insensitive' proteinases in complex extracts. Alternatively, protein extracts are preincubated with specific reversible i nhibitors before electrophoresis, allowing a rapid discrimination of strong and weak interactions implicating proteinases and reversible inhibitors. I n comparison with the standard gelatin/PAGE procedure, that involves copoly merization of gelatin with acrylamide in the resolving gel, this new proced ure simplifies proteinase patterns, avoids overestimation of proteinase num bers in complex extracts, and allows in certain conditions the estimation o f proteinase molecular weights. Stem bromelain (EC 3.4.22.32), bovine tryps in (EC 3.4.21.4), papain (EG 3.4.22.2), and the extracellular (digestive) c ysteine proteinases of five herbivorous pests are used as model enzymes to illustrate the usefulness of this approach in detecting proteinases and in studying their interactions with specific proteinaceous inhibitors potentia lly useful in biotechnology.