Trypsin inhibition

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%. 

Trypsin inhibition activity was determined by measuring the decrease of trypsin activity in the presence of Allium extracts. The results obtained with different types of extracts (bulb, stalk, green leaf-0.5 g/iuL) are presented in Fig. 41.2. All determinations were carried out in triplicate. The higher inhibition activity was obtained with red onion bulb extract (18.55 0.14 Ul/min or 27.94 0.21 U/g fresh plants). Onion bulb extract shows also a high trypsin inhibition (18.48 0.035 UFmin, 27.83 0.053U/g), and all extracts of garlic (bulb, leaf and stalk) show activities between 11.05 1.615 - 13.185 0.205 Ul/min (16.64 2.42 - 19.86 0.31 U/g). 

Akiyama, Y., et al. 1996. Novel peroral dosage forms with protease inhibitory activities. 2. Design of fast dissolving poly(acrylate) and controlled dmg-releasing capsule formulations with trypsin inhibiting properties. Int J Pharm 138 13. 

Acid chloride handles on various templates 49-51 were acylated with equimolar mixtures of 19 different appropriately protected amines to prepare non-peptidic polyamide libraries. The three templates possess different types of symmetry and offer varying degrees of 3D spatial diversity. Screening for trypsin inhibition employing a chromogenic assay identified the xanthane analog 52 (K, = 9.4 pM) as a moderately active, but structurally novel, inhibitor of trypsin (see Fig. 7) [37]. 

Chow MM, Edgar F, Meyer J, Bode W, Kam C-M, Radhakrishnan R, Vijayalakshmi J, Powers JC (1990) The 2.2-A resolution X-ray crystal structure of the complex of trypsin inhibited by 4-chloro-3-ethoxy-7-guanidinoisocoumarin a proposed model of the thrombin-inhibitor complex. J Am Chem Soc 112 7183-7189... 

This approach was reported by Carell et al. [30] and, whilst it has often been cited with the name subtractive deconvolution, it could be also named tailored deconvolution. One of the libraries deconvoluted by this method, and targeted to trypsin inhibition, was prepared in solution adding equimolar amounts of 19 amino acids (Gin excluded) to a tetrafunctional xanthene scaffold, to produce a solution library composed of 65,341 individuals as a single pool (Figure 8.7). The extensive chemical and analytical assessment which assured the quality of the library included synthesis of six smaller model libraries (30-60 compounds), using simplified xanthene scaffolds, and their thorough MS characterization. Further details are not presented here, being out of the purpose of this review, but the conclusions about the library quality can be safely accepted. 

For enzyme inhibition assays, urine is the preferred specimen [4]. Interestingly, Bik can be measured by the inhibition of trypsin in urine but not in plasma. Urinary Bik analysis may also be performed by antibody staining, latex agglutination, and radioimmunoassay (RIA) [4]. Despite the analytical approach used, all Bik forms are measured together. The enzyme inhibition method involves adding known amounts of trypsin to the specimen and monitoring trypsin inhibition. Trypsin activity is assessed by detection of by-products from a cleavable substrate. Dipstick methods are available for the rapid detection of trypsin inhibitors in urine [15, 17 19]. 

G Naray-Szabo (1984) Quantum chemical calculation of the enzyme ligand interaction energy for trypsin inhibition by benzamidines, J Am Chem Soc 106( 16 ) 4584—4589... 

A population of 24 compounds was randomly chosen from the 64 million possible hexapeptides and optimised for trypsin inhibition using a genetic algorithm [21], Biological testing was performed with a chromogenic assay... 

Yokobayashi, Y., Ikebukuro, K., McNiven, S. and Karube, I. Directed evolution of trypsin inhibiting peptides using a genetic algorithm. J. Chem. Soc., Perkin Trans. I, 1996,2435-2437. 

Figure 8. Reformation of disulfides and inhibitory activities on reoxidation of reduced turkey ovomucoid, a dual inhibitor of bovine trypsin, and a-chy-motrynsin with nonoverlapping sites. The disulfides of turkey ovomucoid were reduced with mercapto-ethanol and the product purified. Reoxidation was made with protein concentrations of 60 fxg/ml in 0.006M. Tris buffer at pH 8.2 at room temperature. (Turkey ovomucoid contains eight disulfides, all of which were reduced to 16 sulfhydryls in this experiment. Of these 14 were found at the first sampling and five at the time the experiment was discontinued.) A9 Trypsin inhibition O, a-chymotrypsin inhibition , sulfhydryls per mole (33).

E) Trypsin inhibits intestinal motility, so the substrates can be hydrolyzed for longer periods. 

Krishnan, R., Zhang, E., Hakansson, K., Ami, R.K., TuUnsky, A. et al. (1998) Highly selective mechanism-based thrombin inhibitors structures of thrombin and trypsin inhibited with rigid peptidyl aldehydes. Biochem. 37 12094-12103. 

An interesting example of a three-component PEC is the complex obtained by the polymerization of methacrylic acid (MAA) carried out in the presence of chitosan and PEG. Insulin and BSA could be incorporated in the PEC with good efficiency. However, trypsin inhibition efficiency of this PEC was found to be lower than that of carbopol, a reference polymer. 

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