Benzoyl-DL-arginine-p-nitroanilide

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. 

The methods used were acrosin proteolytic activity (APA) assay (with gelatin) and acrosin activity assay with N-a-benzoyl-DL-arginine-p-nitroanilide (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 trypsin hydrolysis of BAPNA is measured spectrophotometrically (405 nm, 8 = 9.96 cm miol" ) [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- -benzoyl-DL-arginine p-nitroanilide (BAPNA) per minute at pH 7.8 and 25°C and one Trypsin Inhibitor Unit (TIU) will decrease the activity of 2 trypsin units by 50%. 

Trypsin, chymotrypsin, and cathepsin C (all from bovine), and the synthetic substrates glycyl-L-phenylalanine p-naphtylamide (GPNA), 4-phenylazo benzyloxy-car-bonyl-pro-leu-gly-pro-D-arg (PZ-peptide), N-benzoyl-L-tyrosine ethyl ester (BTEE), and Na-benzoyl-DL-arginine p-nitroanilide (BAPNA), were purchased from Sigma Chemical Co (St. Louis, MO). Fresh bluefish (Pomatomus saltatrix) and sheephead samples were purchased from a local fish market (Waldman Plus, Montreal, PQ) and kept in iCe until ready for use. 

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