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.     

New chromogenic and fluorogenic substrates for pyrrolidonyl peptidase.

L-Pyroglutamyl derivatives of p-nitroaniline and 7-amino-4-methylcoumarin were synthesized as new sensitive substrates for pyrrolidonyl peptidase (pyrrolidonecarboxylyl peptidase) from Bacillus amyloliquefaciens. Their hydrolyses could be followed by conventional colorimetric and fluorometric procedures; i.e., in terms of the increase in absorbance at 410 nm caused by the liberation of p-nitroaniline and the emission at 440 nm after excitation at 370 nm depending on the liberation of 7-amino-4-methylcoumarin. Values of Km were estimated to be 0.69 mM for anilide substrate and 0.33 mM for methylcoumarin substrate in the pyrrolidonyl peptidase reaction at pH 8.0. The methylcoumarin compound was about one thousand fold more sensitive than the anilide substrate.     

Application of chromogenic substrates to the determination of peptidases in mycobacteria

The problem of whether peptidase activity of mycobacteria is due to more specific enzymes or to a single, rather wide-spectrum, enzyme is approached by the application of definite chromogenic substrates aminoacyl naphthylamides, NA). This application might serve as an additional very important taxonomic tool for this bacterial family. The identification procedure described is simple and yields exact and rapid results. Other methods suggested for the determination of peptidase activity are presented. Lines of further research in the field are discussed.     

A modified procedure for the preparation of insoluble chromogenic substrates for the determination of proteolytic activity

New insoluble chromogenic substrates for the determination of proteolytic activity were prepared by cross-linking gelatin with glutaraldehyde in the presence of congo red or nigrosin. The prepared substrates could detect approximately 0.4 microgram of trypsin per ml. The spontaneous leakage of dyes in water solutions was negligible.     

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.     

New chromogenic substrates for the assay of alpha-amylase and (1 leads to 4)-beta-D-glucanase

New chromogenic substrates have been developed for the quantitative assay of alpha-amylase and (1 leads to 4)-beta-D-glucanase. These were prepared by chemically modifying amylose or cellulose before dyeing, to increase solubility. After dyeing, the substrates were either soluble or could be readily dispersed to form fine, gelatinous suspensions. Assays based on the use of these substrates are sensitive and highly specific for either alpha-amylase or (1 leads to 4)-beta-D-glucanase. The method of preparation can also be applied to obtain substrates for other endo-hydrolases.     

Evaluation of novel chromogenic substrates for the detection of bacterial beta-glucosidase

AIMS: To evaluate three previously unreported substrates for the detection of beta-glucosidase activity in clinically relevant bacteria and to compare their performance with a range of known substrates in an agar medium. METHODS AND RESULTS: The performance of 11 chromogenic beta-glucosidase substrates was compared using 109 Enterobacteriaceae strains, 40 enterococci and 20 strains of Listeria spp. Three previously unreported beta-glucosides were tested including derivatives of alizarin, 3',4'-dihydroxyflavone and 3-hydroxyflavone. These were compared with esculin and beta-glucoside derivatives of 3,4-cyclohexenoesculetin, 8-hydroxyquinoline and five indoxylics. All substrates yielded coloured precipitates upon hydrolysis in agar. Alizarin-beta-D-glucoside was the most sensitive substrate tested and detected beta-glucosidase activity in 72% of Enterobacteriaceae strains and all enterococci and Listeria spp. The two flavone derivatives showed poor sensitivity with Gram-negative bacteria but excellent sensitivity with enterococci and Listeria spp. CONCLUSIONS: Alizarin-beta-d-glucoside is a highly sensitive substrate for detection of bacterial beta-glucosidase and compares favourably with existing substrates. beta-glucosides of 3',4'-dihydroxyflavone and 3-hydroxyflavone are effective substrates for the detection of beta-glucosidase in enterococci and Listeria spp. SIGNIFICANCE AND IMPACT OF THE STUDY: The data presented allow for informed decisions to be made regarding the optimal choice of beta-glucosidase substrate for detection of pathogenic and/or indicator bacteria.     

Sensitive, soluble chromogenic substrates for HIV-1 proteinase

By replacement of the P1' residue in a capsid/nucleocapsid cleavage site mimic with 4-NO2-phenylalanine (Nph), an excellent chromogenic substrate, Lys-Ala-Arg-Val-Leu*Nph-Glu-Ala-Met, for HIV-1 proteinase (kappa cat = 20 s-1, Km = 22 microM) has been prepared. Substitution of the Leu residue in P1 with norleucine, Met, Phe, or Tyr had minimal effects on the kinetic parameters (kappa cat and kappa cat/Km) determined at different pH values, whereas peptides containing Ile or Val in P1 were hydrolyzed extremely slowly. The spectrophotometric assay has been used to characterize the proteinase further with respect to pH dependence, ionic strength dependence, and the effect of competitive inhibitors of various types.     

Simultaneous determination of zymogen activation time and zymogen level using chromogenic substrates in blood coagulation analysis

The amidolytic activities of plasma generated by means of thromboplastin and Ca++, on the one hand, and by means of partial thromboplastin, a contact activator and Ca++, on the other hand, were determined using synthetic, chromogenic factor Xa substrates with low affinity for thrombin (CH3SO2-D-Leu-Gly-Arg-pNA and CH3SO2-D-Nleu-Gly-Arg-pNA). In this way, the activation process by splitting off the p-nitroaniline was followed. Besides the summary detection of factor Xa was obtained after addition of hirudin. During preincubation with partial thromboplastin and contact acti (Actin) in Ca++-free medium, an amidolytic activity so far unidentified was generated that renders evaluation of the activation process difficult. In the test system with partial thromboplastin, factor Xa could not be determined and the thrombin-like activity that can be inhibited by hirudin did not correspond to the amount of prothrombin present in plasma. In contrast, activation of factor X and prothrombin by thromboplastin and Ca++ could be followed and the content of the two zymogenes could be detected simultaneously. In general, under optimized reaction conditions, automated systems might be developed that would provide additional diagnostic information about determination of clotting time, on the one hand, and about quantitative determination of zymogen, on the other hand.     

Diagnosis of increased thrombotic tendency with chromogenic substrates

In 185 patients suffering from increased thrombotic tendency and in 40 healthy individuals, the following parameters were assayed by the aid of chromogenic substrates: antithrombin III, plasminogen, immediate and total antiplasmin. Furthermore, the vascular activator of plasminogen and the spontaneous aggregation of platelets were examined by other methods. Alterations of plasmatic parameters which are likely to cause an increased thrombotic tendency were detected in 10% of all cases. In patients suffering from arterial thrombotic manifestations hyperactive platelets were found in 42%.     

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.     

Enzymatic synthesis of chromogenic substrates for Glu,Asp-specific proteinases.

Glu,Asp-specific endopeptidases represent a new subfamily of chymotrypsin-like proteolytic enzymes. These enzymes prefer Glu or Asp residues in the P1 position of the substrates. p-Nitroanilides of N-acylated di-, tri- and tetrapeptides with C-terminal glutamic or aspartic acid residues have been obtained. Acyl peptide p-nitroanilides were synthesized via acylation of glutamic or aspartic acid p-nitroanilides using methyl esters of the respective N-acylated peptides, generally with good yields. The reactions were performed in organic solvents using subtilisin 72 sorbed on silica as a catalyst. The kinetic parameters for the hydrolysis of these p-nitroanilides with proteinases from Bacillus intermedius and Bacillus licheniformis were determined.     

Synthesis and characterisation of novel chromogenic substrates for human pancreatic alpha-amylase

Derivatives of maltose and maltotriose were chemically synthesised as substrates for human pancreatic alpha-amylases and subjected to kinetic analysis. Rates measured were shown to reflect both hydrolysis and transglycosylation reactions. 4-O-Methylated derivatives of these substrates underwent only hydrolysis, thereby simplifying kinetic analyses. These modified substrates may be used for the detection and kinetic analysis of alpha-amylases, and are useful in rapidly screening for novel alpha-amylase inhibitors and for subsequent kinetic characterisation.     

Novel chromogenic aminopeptidase substrates for the detection and identification of clinically important microorganisms

A series of amino acid derivatives 8–10, 42 and 43 have been prepared as chromogenic enzyme substrates in order to detect aminopeptidase activity in clinically important Gram-negative and Gram-positive bacteria. Enzymatic hydrolysis liberates the amino acid moiety and either a 4-aminophenol or a 4-dialkylaminoaniline derivative which undergoes oxidative coupling with 1-naphthol or a substituted 1-naphthol giving an indophenol dye. Substrates and 1-naphthols were incorporated into an agar-based culture medium and this allowed growth of intensely coloured bacterial colonies based on hydrolysis by specific enzymes. Red/pink coloured colonies were produced by the substrates 8–10 and blue coloured colonies were formed by the substrates 42 and 43. The l-alanyl aminopeptidase substrates 8 targeted l-alanyl aminopeptidase activity and gave coloured colonies with a range of Gram-negative bacteria. Substrates 9 targeted β-alanyl aminopeptidase activity and generated coloured colonies with selected Gram-negative species including Pseudomonas aeruginosa. Three substrates for l-pyroglutamyl acid aminopeptidase (10a, 10c and 43) were hydrolysed by enterococci and Streptococcus pyogenes to generate coloured colonies. Two yeasts were also included in the study, but they did not produce coloured colonies with any of the substrates examined.     

Spectrophotometric assay for protease activity in ionic liquids using chromogenic substrates

A new spectrophotometric assay has been developed to evaluate protease activity in ionic liquids (ILs). The assay consists of two strategies to enable real-time spectrometric analysis of enzymatic reaction in ILs. First, enzymes are modified with a comb-shaped poly(ethylene glycol), PM₁₃, to obtain a transparent enzyme solution in IL. Second, a chromogenic substrate is used to follow the enzymatic reaction in IL. p-Nitroaniline-derivatized substrates are subjected to protease-catalyzed alcoholysis to release chromogenic p-nitroaniline that can be quantitatively detected by a UV-Vis spectrophotometer. By using this method, we can evaluate protease activity in ILs quite easily without separation of products from the reaction mixture. The availability of the novel assay system was demonstrated in a kinetic analysis of subtilisin-catalyzed reaction in the IL 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ([Emim][Tf₂N]) under different reaction conditions. Because two different serine proteases, subtilisin and α-chymotrypsin, substantially retained its original substrate specificity in the IL, the assay can be extended to other enzymes by using suitable chromogenic substrates.     

The use of chromogenic reference substrates for the kinetic analysis of penicillin acylases.

Determination of kinetic parameters of penicillin acylases for phenylacetylated compounds is complicated due to the low K(m) values for these substrates, the lack of a spectroscopic signal, and the strong product inhibition by phenylacetic acid. To overcome these difficulties, a spectrophotometric method was developed, with which kinetic parameters could be determined by measuring the effects on the hydrolysis of the chromogenic reference substrate 2-nitro-5-[(phenylacetyl)amino]benzoic acid (NIPAB). To that end, spectrophotometric progress curves with NIPAB in the absence and presence of the phenylacetylated substrates and their products were measured and analyzed by numerical fitting to the appropriate equations for competing substrates with product inhibition. This analysis yielded kinetic constants for phenylacetylated substrates such as penicillin G, which are in close agreement with those obtained in independent initial velocity experiments. Using NIPAB analogs with lower k(cat)/K(m) values, kinetic parameters for the hydrolysis of cephalexin and penicillin V were determined. This method was suitable for determining the kinetic constants of penicillin acylases in periplasmic extracts from Escherichia coli, Alcaligenes faecalis, and Kluyvera citrophila. The use of chromogenic reference substrates thus appears to be a rapid and reliable method for determining kinetic constants with various substrates and enzymes.     

Inhibition of the peroxidase-catalyzed oxidation of chromogenic substrates by alkyl-substituted diphenols

A comparative kinetic study of the peroxidase oxidation of three chromogenic substrates--2,2'-azino-bis(3-ethyl-2,3-dihydrobenzothiazoline-6-sulfonic acid), o-phenylenediamine (PDA), and 3,3',5,5'-tetramethylbenzidine--inhibited by trimethylhydroquinone and six tert-butylated pyrocatechols (InH) was carried out at 20 degrees C in 0.015 M phosphate-citrate buffer (pH 6.0) containing organic cosolvents (0-10% ethanol or DMF). The inhibitors were quantitatively characterized by the inhibition constants (Ki), the duration of the lag period in the oxidation product formation (delta tau), and the stoichiometric coefficient of inhibition that specifies the number of radicals terminated by one InH molecule (f). The inhibition could be competitive, noncompetitive, mixed, or uncompetitive, which depended on the nature and structure of the chromogenic substrate-diatomic phenol pair. Various substrate-diatomic phenol pairs exhibited Ki values within the range of 11-240 microM and f values from 0.7 to 2.6. The absence of a lag period was characteristic of oxidation of the substituted o-phenylenediamine-substituted pyrocatechol. The total kinetic parameters and properties of the components allowed us to suggest six chromogenic substrate-substituted diatomic phenol pairs for use in test systems for the determination of antioxidant activity in human body fluids, natural biological preparations, and food. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.     

An inexpensive insoluble chromogenic substrate for the determination of proteolytic activity

Summary A new insoluble chromogenic substrate for the determination of proteolytic activity was developed. This substrate was prepared by incorporating black drawing ink into casein and heating this complex at 200°C for 4 h. It is especially suitable for determining the activity of alkaline bacterial proteinases.