A new approach to the general detection and measurement of proteinase and proteinase inhibitor activities

Abstract

A novel and highly sensitive system for the detection and measurement of proteinase and proteinase-inhibitor activities is described. The method consists of immobilising substrate protein which has a rather random structure and hence is highly susceptible to proteolysis (e.g., casein, denatured haemoglobin, etc.) to an insoluble particle (e.g., Sepharose) and then linking to it a reporter enzyme. The identity of this reporter enzyme is unimportant but should be chosen on grounds of convenience, cost and absence from the samples to be assayed. Globular proteins such as the reporter enzyme are relatively more resistant to proteolysis than the substrate proteins, so the action of the proteinases is to cleave the substrate and release into the supernatant the reporting enzyme. During subsequent incubation this enzyme transforms many hundreds of molecules of its own substrate, resulting in a very substantial amplification factor over the initial peptide bond cleavage. The method is thus far more sensitive than those employing a passive dye label attached to substrate proteins. Using complexes of β-glucosidase linked with glutaraldehyde or m-maleimidobenzoyl N-hydroxysuccinimide ester to casein bound to Sepharose 4B by the CNBr procedure, we have found the approach to be applicable to a wide range of different proteinases and proteinase inhibitors, with a sensitivity limit better than 10 −10g/ml for trypsin and chymotrypsin and 10 −8g/ml for plasmin and bromelain. Measurements have been made of proteinase activity in gel filtration and ion-exchange chromatography fractions of normal and mastitic bovine milks and of trypsin inhibitors in gel filtration fractions of bovine blood serum and whey. On the basis of our results it is suggested that the method may be of general applicability to situations where it is desirable to measure very low levels of such activities in extracts or fluids of biological, biochemical or clinical interest.