Benzoyl-trypsin

Of special interest is the reaction of an acyl enzyme with active-site-directed inhibitors in comparison with the free enzyme. Therefore, we studied the hydrolysis of the substrate Bz-Arg-pNA by trypsin and benzoyl-trypsin in the presence of the naturally occurring inhibitor aptotinin [21]. The substrate is cleft... 

Figure 4 Reaction of trypsin (T) and benzoyl-trypsin (BT) (0.1 pmol/L) with synthetic substrate (Bz-Arg-pNA) in the absence (dotted line) or presence (solid line) of the inhibitor aprotinin. (From Ref. 21.)...

The parameters are usually obtained from a series of initial rate experiments performed at various substrate concentrations. Data for the hydrolysis of benzoyl-L-tyrosine ethyl ester (BTEE) by trypsin at 30 V and pH 7.5 are given below ... 

Wang, S.S., and Carpenter, F.H. (1968) Kinetic studies at high pH of the trypsin-catalyzed hydrolysis of Na-benzoyl derivatives of L-arginamide, L-lysinamide, and S-2-aminoethyl-L-cysteinamide and related compounds./. Biol. Chem. 243, 3702-3710. 

Fig. 8. Effect of linear flow velocity of an L-benzoyl arginine ethylester solution (0.2 mol/1) on the enzymatic activity of trypsin immobilized on poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads (curve 1) and monolith (curve 2) (Reprinted with permission from [90]. Copyright 1996 Wiley-VCH). Reactor 50 mm x 8 mm i.d., temperature 25 °C...

The methods used were acrosin proteolytic activity (APA) assay (with gelatin) and acrosin activity assay with N-a-benzoyl-DL-arginine-p-nitroanihde (BAPNA)-Triton X 100, and BAPNA assay for trypsin activity [9-11]. The antioxidant activity was tested spectrometrical with ABTS+ [12]. Cytotoxicity of extracts was determined by MTT Cell Proliferation Assay [13]. 

Trypsin inhibition The ability of the various trypsin inhibitors Allium extracts) to prevent tiypsin hydrolysis of BAPNA is measured spectrophotometrically (405 nm, = 9.96 cm pmoT ) [11] at 25°C, with a Jasco UV-Vis spectrometer by time course measurement of AAbs. One trypsin unit hydrolyzes 1.0 pmol of A-ft-benzoyl-DL-arginine /i-nitroanilide (BAPNA) per minnte at pH 7.8 and 25°C and one Trypsin Inhibitor Unit (TIU) will decrease the activity of 2 trypsin units by 50%. 

Catalytic tests were performed in a glass vessel equipped with a stirrer motor. Two monoliths (diameter 4.3 cm, length 4 cm) were mounted in plane on the stirrer axis. The total reaction volume was 2.5 1. Lipase was assayed in the acylation of vinyl acetate with butanol in toluene. Initial reaction rate was followed by GC analysis. Immobilized trypsin was used in the hydrolysis of N-benzoyl-l-arginine ethyl ester (BAEE) in a 0.01 M phosphate buffer pH 8 at 308 K. The reaction was followed by UV-VIS at 253 nm, and reaction rate was calculated in the mass transfer limited situation. 

FIGURE 11.5 Degradation of N-a-benzoyl-l-arginine ethyl ester by trypsin. All three carboxyl containing polymers have antitrypsin activity, but the activity of trypsin in the presence poly(ethylene glycol) modified poly(methacrylic acid) is not greatly reduced. (Adapted from Madsen and Peppas 1999.)... 

A substrate L-benzoyl arginine /)-nitroanilide hydrochloride was hydrolyzed by trypsin, with inhibitor concentrations of 0, 0.3, and 0.6 mmol 1 T The hydrolysis rates obtained are listed in Table 3.3 [5]. Determine the inhibition mechanism and the kinetic parameters (A", and /Cj) of this enzyme... 

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

In Table II are shown the results from kinetic studies with commercially available gastric and pancreatic enzymes. Trypsin was strongly inhibited, at least at a low concentration of casein as substrate. The hydrolysis of benzoyl arginine ethyl ester (BAEE) by trypsin was non-competitively inhibited, giving a 30% reduction of Vmax at 0.5 mg/ml of the LMW fraction. Carboxypepti-dase A, and to a lesser extent carboxypeptidase B, were non-competitively inhibited as well. Pepsin and chymotrypsin were not affected by the conditions used in these assays. 

Fig. 4.4 Increase of p-nitroaniline in a solution containing the trypsin substrate benzoyl-arginine-p-nitroanilide and trypsin at 37°C and phosphate buffer pH 7.6 -o- buffer only, - -0.1% chitosan, -A- 0.025% chitosan-aprotinin,...

Substrate Solution Dissolve 85.7 mg of A-benzoyl-l-argi-nine ethyl ester hydrochloride, suitable for use in assaying trypsin, in sufficient water to make 100 mL. 

Trypsin (5 xg/ml), type IX from porcine pancreas, and 0.375 mM A-a-benzoyl-L-arginine-p-nitroanilide (l-BAPNA) were added to a 96-well assay plate containing... 

We may also mention that peptide-bond absorption at somewhat longer wavelengths has been employed by Mitz and Schleuter (1957), and by Schmitt and Seibert (1961) to estimate the rates of digestion by peptidases acting on simple peptides. Schwert and Takenaka (1955) describe difference-spectral methods for assaying chymotrypsin and trypsin with acetyl-L-tyrosine ethyl ester and benzoyl-L-arginine ethyl ester as the respective substrates. 

The specificity of chymotrypsin for hydrolysis of peptide bonds formed by the carbo,xyl groups of tyrosine, phenylalanine, and tryptophan has been recognized for some time (Green and Neurath, 1954 Desnuelle, 1960). Action on synthetic substrates of leucine (Goldenberg et al., 1951) and methionine (Kaufman and Neurath, 1949) also has been noted although at much slower rates than observed with the aromatic amino acid derivatives. When protein substrates or synthetic ester substrates are examined, it is evident that a variety of bonds can be hydrolyzed by chymotrypsin. Inagami and Sturtevant (1960) observed that chymotryptic hydrolysis of a-benzoyl-L-arginine ethyl ester, a typical trypsin substrate, occurred at a maximum rate which was 20% of that observed with trypsin. Several ester substrates, such as p-nitrophenylacetate (Hartley and Kilby, 1954), are also hydrolyzed. 

Various enzymes, lysozyme, catalase, phosphatase, malate/formate dehydrogenase, adenylase kinase, pymvate decarboxylase, alkaline phospatase, serum albumin, lipoprotein, interferon, growth factor, polypeptide hormone DNA, RNA, y-globulin, proteinase, carboxypeptidase, endotoxins, pyrogen, IgG, trypsin inhibitor, transferrin, casein, L-benzoyl arginine ethyl ester, cytocrome C IgG, heparin... 

AAT can be quantified by all immunochemical methods, with immunoturbidimetry and immunonephelometry the most commonly used methods. Because it constitutes about 90% of the serum inhibition of trypsin or elastase activity against small substrates, such as benzoyl-DL-arginine p-nitroanilide, it can also be semiquantified by the inhibitory capacity of serum for these enzymes however, this assay is not specific for AAT. 

Table III also shows that trypsln-cvb-hydrogel derivatives retain in the range of 11-45% the original enzyme activity, which is about average for the covalently bound trypsin on polymers containing hydrogel. For comparison, Mosbach (2.) immobilized trypsin on a crosslinked copolymer of acrylamide and hydroxyethyl-methacrylate via CNBr coupling, and analyzed it photometrically with a- -benzoyl-Dl-arginine-p-nltroanlllde (BAPNA), reporting a 35% retention of activity. o Driscoll and co-workers (1 ) however, reported an equivalent of 1.3% efficiency when trypsin was physically entrapped in a crosslinked PHEMA gel and assayed with TAME.

---