Detection of proteolytic enzymes in polyacrylamide gels

Aminopeptidases (1), dipeptidyl aminopeptidases (2), pyrrolidonyl peptidases (3), and carboxypeptidases (4,5) can be detected in polyacrylamide gels with appropriate-β-naphthylamide or carbonaphthoxyamino acid substrates while dipeptidases, tripeptidases (6), carboxypeptidases (7), and aminopeptidases can be detected by the coupled l-amino acid oxidase-peroxidase method of Lewis and Harris (6).In contrast, fewer methods are available for the detection of proteinases in gels. Trypsin-like (8,9) and chymotrypsin-like (5,10) proteinases can be detected with chromogenic β-naphthylamide and β-naphthol ester substrates, but proteinases such as thermolysin (11) and other bacterial neutral metal chelator-sensitive proteinases (12) cannot. For these latter proteinases, whose specificities are directed towards the amino acid residue containing the amino group of the bond to be hydrolyzed, and for proteinases, whose specificities remain to be determined, other methods of detection have to be employed.Uriel and Avrameas (13) detected proteinases in agarose gels by overlaying these gels with a second agarose gel mixture containing the substrate and a suitable pH indicator. However, the method suffers from interference by gel buffers and the instability of the pattern developed. Another procedure is to bring the gel in contact with a gelatinous layer of film material (14,15). This has been done successfully with tissue sections (16), paper electrophoretograms (17) and agarose gel separations (18).The most suitable approach is to diffuse an appropriate protein substrate into the gel after electrophoresis and detect the proteinase activity directly. Several variations of this method have been published (19–22), each with its own advantages and disadvantages. In this report a simple, sensitive method using cytochrome c as substrate, and requiring no staining, is described. This report describes its application to the detection of thermolysin and trypsin in anionic and cationic gel systems, respectively. The method has also been routinely used to locate bacterial and insect proteinases after electrophoresis.     

Substrate-containing gel electrophoresis: sensitive detection of amylolytic, nucleolytic, and proteolytic enzymes

Electrophoresis of hydrolytic enzymes under nondenaturing conditions on acrylamide gels containing the appropriate high-molecular-weight substrates entrapped on the gel has been explored as a general method for sensitive enzyme resolution and detection. Under electrophoresis conditions of optimal enzyme activity, the enzymes may bind tightly to the fixed substrate and can only migrate in the electrophoretic field as the substrate is hydrolyzed. When the gels after electrophoresis in this “binding mode” are stained with substrate-detecting reagents, clear tracks of enzyme migration are observed, and the length of each track is a function of the amount of enzyme present in that track. Multiple forms of a given enzyme activity have not been and are not likely to be observed under these conditions. Under electrophoresis conditions of minimal (or suboptimal) enzyme activity, the enzymes do not bind to the fixed substrate and their mobility in the electrophoretic field does not appear to be significantly affected by the presence of substrate. After electrophoresis in this “nonbinding mode” the gels are incubated under conditions of optimal enzyme activity to allow substrate hydrolysis to take place before they are stained with substrate-detecting reagents, and active enzymes are detected as clear bands. Multiple forms of a given activity which were resolved during electrophoresis in the nonbinding mode are reflected by the presence of individual bands. The substrate-containing gel electrophoresis technique does not appear to be amenable to precise quantification of enzymes. By comparing the length of the clear tracks or the degree of staining of the activity bands for a range of enzyme concentrations, however, it is possible to establish the smallest amount of enzyme that can unequivocally be detected under a given set of conditions; from such studies we estimate that the sensitivity of detection with the substrate-containing gel electrophoresis technique can be orders of magnitude better than that obtained with other methods. The levels of detection observed in the work presented here were about 50 pg for α-amylase run on starch-containing gels, 1 pg to 1 ng for nucleases run on DNA- or RNA-containing gels, and 100 pg to 10 ng for 11 different pure and crude protease preparations run on gels containing heat-denatured bovine serum albumin.     

New proteolytic enzymes in yeast

Yeast mutants lacking three proteolytic enzymes—proteinase B, carboxypeptidase Y, and carboxypeptidase S—have been constructed. Search for new proteolytic activities in these mutants with the aid of chromogenic peptide substrates developed for serum proteinases led to the detection of new proteolytic activities, active in the neutral pH range. Sephadex chromatography of a 100,000g supernate of mutant extracts, tests against four different substrates, and partial characterization of their sensitivity to various inhibitors indicate multiple activities. Two activities, called proteinase M and proteinase P, were found in the sedimentable membranous fraction of mutant extracts.     

Stabilization of proteolytic enzymes in solution

The stability of the neutral and alkaline proteases in a Bacillus subtilis enzyme mixture was studied in aqueous solutions at room temperature. Stabilization of the proteases in solution for periods up to 25 days was achieved by the addition of various protein preparations including casein and soya protein. The degree of stabilization by casein was concentration dependent to about 2% protein. The instability of the neutral protease in solutions of the B. subtilis enzyme mixture was shown to be due primarily to proteolysis by the alkaline protease since the diisopropylfluorophosphate-treated enzyme was quite stable. Formulation of such enzyme solutions at low pH gave greater stability as did solutions containing an alkaline protease inhibitor from potatoes. A Conceptual approach to the formulation of enzyme solutions containing proteolytic enzyme to ensure maximum stability is proposed.     

The versatility of proteolytic enzymes

The growing realization of their physiological importance has generated renewed interest in the study of proteolytic enzymes. Modern methods of protein chemistry and molecular biology have revealed new insights into the protein and gene structure of a variety of protein precursors and their processing by limited proteolysis. Examples are given in this review for transmembrane processes and the role of signal peptidases of both eukaryotic and prokaryotic origin, the processing of prohormones and precursors of growth factors, protein components of blood coagulation, fibrinolysis, and of the complement system, and a group of granulocyte proteases, including the mast cell serine proteases. The relationship of homologous domains found in many of these proteases and their zymogens to protein evolution is a recurrent theme of this discussion.     

Inactivation of proteolytic enzymes by cestodes

Inhibitor activity of cestodes from intestines of different hosts (sea birds, salt-water fish, and freshwater fish) was investigated. Alcataenia larina, Arctotaenia tetrabothrioides, Tetrabothrius erostris, T. minor, Wardium cirrosa, Bothriocephalus scorpii, Eubothrium rugosum, and Triaenophorus nodulosus were able to inhibit the activity of the commercial trypsin and activity of proteinase homogenates of the intestinal mucosa of the hosts. It was suggested that the inhibitor produced by the cestodes is specific for trypsin and protects them from the digestive enzymes of the host.