P-Nitroanilide

The GMT in human serum reacts most rapidly with Y-glutamyl-p-nitroanilide at pH 8.2. The same activity is found in 2-amino-2-methylpropane-l 3 diol, diethanolamine, triethanolamine and tris buffers. Magnesium ions have no effect on the activity but favor the solubilization of the substrate. Bondar and Moss (54) found that free glutamate, due to elevated serum glutamate concentrations or glutamate released by substrate breakdown, increases the apparent GMT activity. They concluded that the assay should be performed in the presence of 1.0 vM/1 glutamate in order to reduce the possibility of falsely elevated results. This was not observed by others. Rowe and co-workers have indicated that certain batches of p-nitroanilide substrate contain impurities which may reduce GMT activity and increase the values ( ). Huesby and Stromme (56) confirmed the presence of such impurities and recommended pyridine extraction for substrate purification. 

Abbreviations ADA, adenosine deaminse BAEE, N,a-benzoyl-L-arginine ethyl ester, BTEE, N,oc-benzoyl-L-tyrosine ethyl ester BTNA, N,a-benzoyl-L-tyrosine-p-nitroanilide ZAPA, N,a-benzoylcarbonyl-L-arginine-p-nitroanilide. 

The thermodynamic activation parameters for the enzyme-catalysed reaction are very different from those for the uncatalysed process (Albers et at., 1990). For the isomerization of succinyl-alanyl-leucyl-prolylphenylalanyl-p-nitroanilide catalysed by recombinant human FK binding protein AH = 5.85 kcal mol"1 and AS = -44. e.u. This compares with figures of 18.9 kcal mol 1 and — l.lbe.u. for the uncatalysed reaction of the same substrate. Probably a different step is rate determining in the enzyme-catalysed reaction. 

The substrate used was the synthetic peptide N-Succinyl-Alanine-Alanine-Proline-Phenylalanine p-Nitroanilide which upon hydrolysis liberates 4-Nitro-aniline which is conveniently measured at 410 nm. 

FIGURE 8.5 Synthesis of p-nitroanilides by aminolysis (A) of the mixed anhydride obtained with M = A-methylmorpholine as base,14 (B) of the dichlorophosphoric anhydride obtained with P = pyridine as base and solvent, [Rijkers et al., 1991] and (C) by reaction with p-nitrophenylisocyanate. (Nishi Noguchi, 1973). 

DTS Rijkers, HC Hemker, GHL Nefkens, GI Tesser. A generally applicable synthesis of amino acid p-nitroanilides as synthons, in CH Schneider, AN Eberle, eds. Peptides 1992. Proceedings of the 22nd European Peptide Symposium. Escom, Leiden, 1993, pp 175-176. 

The total cDNA construct of NPN43C9 was expressed efficiently in E. coli cells and protein purified, and its catalytic properties were assessed in both the monoclonal antibody and the single-chain antibody (scFv) 7A4-1/212 for the hydrolysis of p-nitroanilide [50] and the related p-chlorophenyl ester [51a] (Fig. 19). Virtually identical kca, and Km values were obtained for both 7A4-1/212 and NPN43C9. This activity was decreased in both cases by the addition of the inhibitor w-nitroanilide [52], which gave K = 800 p.M and 400 pM for the NPN43C9 and 7A4-1/212, respectively. 

Supported reagents have found application in many areas of synthesis including the construction of small peptides, the traditional foundation stone of solid phase synthesis. For example a recent paper describes the preparation of dipeptide p-nitroanilide and phosphonate libraries by supported carbodiimide coupling and scavenger purification (Scheme 2.52) [79]. 

The a-chymotrypsin-catalyzed hydrolysis of the p-nitroanilide group in acrylamide copolymers with monomer I proceeds with a rate comparable to that of the monomer... 

ELISA has been used for measuring caspase activity. For the ELISA of intracellular caspase activity at the very early stages of apoptosis, apoptotic cells are first lysed to isolate their intracellular contents. Different caspase activities in the cell lysate can then be determined by the addition of a caspase-specific tetrapeptide substrate that is conjugated to the color reporter molecule p-nitroanilide (pNA) (e.g., DEVD-pNA for caspase-3 and lETD-pNA for caspase-8). The cleavage of the substrate peptide by the caspase releases the chromophore pNA, which can be... 

The influence of the structure of the main chain on the chymotrypsin catalyzed release of p-nitroaniline is shown in Fig. 15. Oligopeptide p-nitroanili-des were attached to HPMA copolymers (as side-chains) and to polyethylene glycol (end-point attachment). From the values of kcat/KM it is evident that all oligopeptide p-nitroanilides attached to PEG were cleaved faster than those attached to HPMA copolymers. It appeared that the PEG substrates fit better into the active site of chymotrypsin due to the linearity and flexibility of the PEG molecule, and the type of spacer attachments [255]. 

Table 3.3 Hydrolysis rate of L-benzoyl arginine p-nitroanilide hydrochloride by trypsin with and without an inhibitor.

A straightforward approach is to hunt for short polypeptides that meet the specificity requirement of an enzyme but which, because of peculiarities of the sequence, are acted upon very slowly. Such a peptide may contain unusual or chemically modified amino acids. For example, the peptide Thr-Pro-nVal-NMeLeu-Tyr-Thr (nVal=norvaline NMeLeu = N-methylleucine) is a very slow elastase substrate whose binding can be studied by X-ray diffraction and NMR spectroscopy.6 Thiol proteases are inhibited by succinyl-Gln-Val-Val-Ala-Ala-p-nitroanilide, which includes a sequence common to a number of naturally occurring peptide inhibitors called cystatins.f They are found in various animal tissues where they inhibit cysteine proteases. 

Wild-type subtilisin BPN with the mutation Ser — Cys-24 has a kcat value of 59 s 1 and a KM value of 200 fxM with the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide, compared with a rate constant of 1.1 X 10 8 s 1 for its spontaneous hydrolysis under the same conditions. Replacement of Asp-32, His-64, and Ser-221 one at a time by alanine reduced the value of kcat by factors of 3 X 104, 2 X 106, and 2 X 106, respectively. Converting all three to alanine also decreases activity by 2 X 106. The value of KM increases only by a factor of two on all these mutations.34 It is unlikely that the residual activity results from the presence of a small amount of wild-type active site in the thiol mutants... 

Substrates of the form succinyl-X-Ala-Pro-Phe p-nitroanilide, where P4 (= X) is a hydrophobic residue, have similar values of kcJKM. The residue at the bottom of the S4 site is a large hydrophobic, lie-107. Mutating lie — Gly-107 to increase the size of the pocket decreases kcat/KM for X = Ala some 200-fold, with only small effects for X = Phe, lie, or Leu. The larger side chains complement the lesion caused by introducing a cavity in the protein.38,39... 

Dissolve 78.1 mg Suc-AlaAlaProPhe-p-nitroanilide (Bachem, Enzyme Systems Products, or Sigma) in 10 ml dimethyl sulfoxide or dimethylformamide (12.5 mM). Store up to several months at 4°C, but check for the appearance of yellow color before use. 

Table V gives data on the specificity of the purified y-glutamyl cyclotransferases obtained from human and sheep brain the two enzymes exhibited similar specificity. The model substrate, L-y-glutamyl-L-y-glu-tamyl-p-nitroanilide was the most active substrate the corresponding l,d-, d,l-, and D,D-isomers were inactive. The enzyme preparations also...

Standardization p-nitroanilide of myri stic acid Calibration hop extract Same as ASBC... 

A. A Continuous Colorimetric Assay Using p-Nitroanilide Peptides... 

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