Advantages of orange-labelled collagen and gelatine as substrates for rapid collagenase activity measurement.

A simple method for the determination of collagenase activity utilising dye-labelled substrates is proposed. It consists in labelling gelatine and bovine Achilles tendon collagen under nondenaturing conditions with the dye active orange GT. As verified with two different enzyme preparations, labelling did not dramatically change the susceptibility to collagenases. The kinetic parameters obtained for dye-labelled collagen and gelatine were compared to those obtained for the hydrolysis of native insoluble collagen (Mandl's method). The method offers the following advantages: it is rapid, reproducible, does not require special equipment and is more specific for collagenases than the widely used azocoll and Mandl methods.

N-anthraniloyl-Ala-Ala-Phe-4-nitroanilide, a highly sensitive substrate for subtilisins.

A new substrate for subtilisins, anthraniloyl-Ala-Ala-Phe-4-nitroanilide, has been synthesized and characterized. The peptide is a fluorogenic substrate that is intramolecularly quenched without loss of its chromogenic properties and offers a possibility for double-assay kinetic analysis. The kinetic parameters determined for subtilisin Carlsberg are Km = 0.004 mM, kcat = 104 s-1, and those for subtilisin BPN' are Km = 0.020 mM, kcat = 49 s-1. The substrate is extremely sensitive for subtilisins; the specificity constants are 10-fold higher than the corresponding values for the widely used substrate, succinyl-Ala-Ala-Pro-Phe-4-nitroanilide, and 200- to 1000-fold higher than the values obtained with succinyl-Ala-Ala-Phe-4-nitroanilide. The favorable effect of the anthraniloyl group as a P4 residue in the substrate sequence Ala-Ala-Phe-4-nitroanilide was assumed to be due to an ability to stiffen S4-P4 interactions. The mechanism proposed is hydrogen bond formation between the phenol group of tyrosine-104 and the amino group of the anthraniloyl moiety. In the spectrophotometric assay with the new substrate, the lower detection limit for subtilisin Carlsberg was 1 nM.

An express immunological method for detection of human seminal plasma.

Monoclonal antibodies (Mabs) against human seminal plasma (HSP) were produced and during screening procedures dissociation constants of the antigen/antibody complexes were determined. Mab 1E5 was selected for further studies because of its high reactivity in an enzyme-linked immunoassay (ELISA) and high affinity for its corresponding antigen. The specificity of Mab 1E5 was checked in absorption ELISA with human organ extracts and some biological secretions. It was established that the 1E5-corresponding epitope was a thermostable peptide moiety which could be detected in HSP, only. This monoclonal antibody was used for the development of an express method for detection of human semen. The assay was applied for screening of 57 cases of suspected rape. A complete correlation was found between the results obtained by the proposed test and by routine microscopic methods. The newly designed immunoassay is reliable, it is easily performed and it is less time-consuming.

Action of urokinase and trypsin on angiotensin, ACTH and the oxidized B chain of insulin.

N-terminal analysis of the products of hydrolysis of angiotensin, ACTH and the oxidized B chain of insulin after 4 h incubation with trypsin and urokinase reveals a great qualitative similarity in the action of the two enzymes. As expected, the rates of hydrolysis differ significantly and are much higher in the case of trypsin catalysis than in the case of urokinase catalysis. Unexpectedly, however, a decrease in the difference between the catalytic activity of the two enzymes, by increasing the number of Arg and Lys residues present in the substrate, has been observed.

Kinetic study of the effect of heparin on the amidase activity of trypsin, plasmin and urokinase.

It was found that the effect of heparin on the amidase activity of urokinase (E C 3.4.21.31), plasmin (E C 3.4.21.7) and trypsin (E C 3.4.21.4) depended on the substrate used. No effect of heparin on the amidase activity of urokinase and trypsin was observed when Pyro Glu-Gly-Arg-p-nitroanilide (S-2444) and alpha-N-acetyl-L-lysine-p-nitroanilide (ALNA) were used as substrates. Heparin acted as a uncompetitive inhibitor of trypsin (Ki = 1.2 X 10(-6) M), plasmin (Ki = 4.9 X 10(-6) M) and urokinase (Ki = 1.0 X 10(-7) M) when Bz-Phe-Val-Arg-p-nitroanilide (S-2160), H-D-Val-Leu-Lys-p-nitroanilide (S-2251) and plasminogen, respectively, were used as substrates. These results, as well as the data obtained by studying the effect of the simultaneous presence of heparin and competitive inhibitors suggest that although heparin is not bound at the active center of these enzymes, it may influence the effectivity of catalysis.

Hydrophobic interactions in the urokinase active centre. Inhibitory action of alkyl ammonium and amidinium ions: comparison with trypsin.

In order to obtain information concerning the binding site of urokinase (EC 3.4.99.26) the inhibitory action of aliphatic ammonium and amidinium ions has been studied. The comparison between the corresponding K1 values for urokinase and trypsin shows that although the two enzymes possess a hydrophobic binding pocket of presumably equal length, they differ mainly in their behaviour towards n-alkylammonium ions with an alkyl chain longer than four methylene groups and towards benzylammonium and amidinium ions. This is an indication of a less rigid binding site in urokinase than in trypsin.