Trypsin catalyzed hydrolysis of new chromogenic arginine substrates

Trypsin catalyzed hydrolysis of seven new chromogenic arginine substrates, N alpha-benzyloxycarbonyl-L-arginine-3-nitro-5X-anilide (X = H, CF3, SO2CH3, F, Cl, Br and I) were studied. These substrates are suitable for studying electronic effects on trypsin activity. The Km and kcat values for the hydrolysis of each substrate were determined and found to differ significantly for the various substrates. The Hammett plot of the catalytic rate constants gave a straight line with a negative rho value (-0.82) thus indicating that electron withdrawing substituents retard the trypsin catalyzed hydrolysis of the new anilide substrates.     

Evaluation of new chromogenic substrates for the detection of coliforms

Aims: To evaluate a new range of chromogenic substrates for the detection of β‐galactosidase activity in coliforms and to compare their performance in agar media and broths. Methods and Results: Sixteen novel galactoside substrates were prepared and incorporated into agar and broth. Their performance was compared using Escherichia coli (five strains), Salmonella (two strains), Enterobacter (two strains), Klebsiella, Pseudomonas, Listeria, Serratia, Shigella, Citrobacter, Proteus and Staphylococcus as well as pathological urine samples. The six substrates out of the initial 16 that showed the greatest sensitivity were VQE‐gal, VQM‐gal, VLPr‐gal, VLE‐gal, VLM‐gal and VBzTM‐gal, whose released chromophores were red, brown or purple. VQE‐gal and VLPr‐gal were studied in greater detail and were incorporated into agar medium. Coliform colonies appeared red and brown respectively, following incubation at 37°C for 24 h; however, positive results were obtained within a working day. The VQE‐gal medium was compared with some commercially available media. Conclusions: The range of substrates described can be used in broths as well as in agars. The VQE agar allows the detection of coliforms within a working day. VQE‐gal medium proved to be more sensitive when compared to other available chromogenic media and allows the unambiguous detection of coliforms.     

Actions of two serine proteases from Trimeresurus jerdonii venom on chromogenic substrates and fibrinogen

Jerdonobin and jerdofibrase are two serine proteases purified from the venom of Trimeresurus jerdonii. The Michaelis constant K(m) and the catalytic rate constant K(cat) of jerdonobin or jerdofibrase on three chromogenic substrates, H-D-Pro-Phe-Arg-pNA (S2302), H-D-Phe-pipecolyl-Arg-pNA (S2238), and H-D-Val-Leu-Lys-pNA (S2251) were obtained from lineweaver-Burk plots. Jerdofibrase could hydrolyze all three substrates, but jerdonobin had no detectable activity on S2251, suggesting a relatively broader substrate specificity for jerdofibrase than jerdonobin. By SDS-PAGE, jerdofibrase preferentially degraded Bbeta-chain of fibrinogen. It also degraded Aalpha-chain of fibrinogen with relatively slow activity, but did not act on the gamma-chain. In contrast, jerdonobin did not degrade fibrinogen within 12 h. Fibrinopeptides liberation test, identified by HPLC, showed jerdonobin released fibrinopeptide A and a small amount of fibrinopeptide B. Unlike jerdonobin, jerdofibrase mainly released fibrinopeptide B. These results indicate that the two enzymes differ in their ability to hydrolyze chromogenic substrates and in their actions on fibrinogen.     

Differentiation of two variants of type-AB GM2-gangliosidosis using chromogenic substrates.

Two variants of type-ABGM2-gangliosidosis can be distinguished by using p-nitrophenyl-6-sulfo-2-acetamido-2-deoxy-beta-D-glucopyranoside (PNP-GlcNAc-6-SO4) as substrate. One of the variants is caused by a deficiency of the activator for the hydrolysis of GM2-ganglioside. The beta-hexosaminidase A from this variant has a normal activity toward both PNP-GlcNAc and PNP-GlcNAc-6-SO4. A second variant caused by a defect in the enzyme, beta-hexosaminidase A, exhibits severely attenuated activity toward PNP-GlcNAc-6-SO4 but normal activity toward PNP-GlcNAc.     

New chromogenic substrates for X-prolyl dipeptidyl-aminopeptidase

We have synthesized several new chromogenic substrates, p-nitroanilides of the dipeptides, Gly-Pro, Ala-Pro, Lys-Pro, Arg-Pro, Glu-Pro, and Asp-Pro, for X-prolyl dipeptidyl-aminopeptidase. These have permitted the development of a simple assay of the enzyme in which p-nitroaniline liberated directly or after the Bratton-Marshall reaction is measured spectrophotometrically. The enzyme activity was measured in human serum or in homogeneous enzyme purified from human submaxillary gland. The homogeneous enzyme hydrolyzed each substrate to produce X-Pro and p-nitroaniline. The optimum pH was at 8.7, except with Arg-Pro p-nitroanilide (8.0). Serum enzyme hydrolyzed Gly-Pro p-nitroanilide to p-nitroaniline and Gly-Pro, which was further hydrolyzed to Gly and Pro by an imidodipeptidase in serum. Gly-Pro β-naphthylamide or Gly-Pro-Leu was a competitive inhibitor with each X-Pro p-nitroanilide as substrate. Gly-Pro p-nitroanilide had the highest activity among the substrates at pH 8.7, followed by p-nitroanilides of Ala, Lys, Arg, Glu, and Asp in a decreasing order of activity.     

Exponential function of chymotrypsin action

Enzyme kinetics are usually described by the hyperbolic Michaelis-Menten equation, but they can also be described by the following exponential function: -dS/dt = Vm [1 - exp (-S/Km)]. The time-dependent decrease of the substrate (-dS/dt) is an exponential function of maximal velocity (Vm), the Michaelis constant (Km) and the actual substrate value (S). This exponential function is based on the assumption that the association of the substrate-enzyme complex is a concentration-dependent process, whereas the transformation of the substrate-enzyme complex is time-dependent. It can be shown that this exponential function is a more general solution of which the hyperbolic Michaelis-Menten equation is a special derivative under the conditions of low substrate (S) and high constant (Km) values. If the association process is time-dependent, the decline in substrate values will show a more concave curve. However, exponential functions in general are more concave than hyperbolic functions. Probably, therefore, the enzyme action of chymotrypsin could be described more appropriately by the present exponential function than by the conventional hyperbolic function.     

Comparison between two chromogenic substrates for revealing an immunoperoxidase reaction of human metaphase chromosomes

To study the binding of an antiadenosine serum to human chromosome DNA, two types of chromogenic reagents were compared. The procedure is as follows: lymphocytes with metaphase chromosomes were spread on slides; some slides were irradiated by UV; all slides were then incubated with antiadenosine rabbit serum and then with antirabbit sheep serum labelled with peroxidase; the latter was revealed in the classical manner by 3,3'-diaminobenzidine (DAB), or, alternatively, by p-phenylenediamine plus pyrocatechol (PPD-PC). The present study shows that the results obtained with PPD-PC were equivalent, if not superior, to those obtained with DAB. In the case of weaker reactions, results with PPD-PC were superior. Furthermore, this reagent has the major advantage of being noncarcinogenic.     

The action of elastase on p-nitroanilide substrates

The action of elastase has been studied on four p-nitroanilides: BOC-(Ala)₂-NA, (Ala)₃-NA, Ac-(Ala)₃-NA and BOC-(Ala)₃-NA. The second order rate constant kcat/Km increases considerably with the chain length of these substrates. With (Ala)₃-NA, activation by excess substrate was observed. DMF and DMSO inhibit strongly the elastase catalyzed hydrolysis of Ac- and BOC-(Ala)₃-NA. The later substrate may be used to assess rapidly elastase activity: concentrations as low as 0.2 μg/ml may be determined accurately.