The chemical modification of pepsin carboxyl groups using N-(2,4-dinitrophenyl)-hexamethylenediamine or compound (I) when measured against hemoglobin and 50 against N-acetyl-L-phenylalanyl-Ltyrosine as substrates. It was established that the modification of three carboxyl groups took place : /?-carboxyl group of the aspartic acid residue, y-carboxyl group of the glutamic acid residue and the a-carboxyl group of C-terminal alanine. It is assumed that this reaction is preceded by the hydrophobic binding at some site, specific for the pepsin hydrophobic zone, with a subsequent reaction of I with the carboxyl groups localized near this zone. Using p-bromophenacyl bromide, Erlanger et al. identified another aspartic carboxyl group which does not belong to the active site, but probably plays some role in the enzyme-substrate binding [6]. We studied the modification of carboxyl groups of pepsin by the reaction with an amine in the presence of a water-soluble carbodiimide. With the purpose of achieving selective modification of this enzyme, we changed the reaction conditions suggested by Hoare and Koshland for total covering of carboxyl groups [7], by the use of sufficiently lower concentrations of both reagents : a water-solublc carbodiimide and a nucleophile. Another feature of the procedure was the application of the coloured aminc N-(2,4-dinitrophenyl)-hexamethylenediamine as the nucleophile. This allows estimation of the incorporation of the amine into the protein directly from the ultraviolet spectrum of the modified protein and significantly simplified the following identification of its attachment site. MATERIALS AND METHODSThe modification of carboxyl groups in chromatographic homogeneous porcine pepsin [8] was carried out a t 20 "C as follows: 100 mg (2.9 pmol) pepsin was dissolved in 100 ml 2.5 mM solution of compound I which contained 20//, (v/v) dimethyl formamide. The solution was adjusted to pH5.5 with aqueous NaOH and 26, 43 or 430 mg (a 21, 33 or a 330-fold excess respectively) .of 1-cyclohexyl-3-12'-(N-meth ylmorpholino) -ethyl] -carbodiimide p-toluenesulphonate (compound 11) was added; pH 5.5 was kept constant by the addition of 0.05N HCl (Radiometer model TTTIc titrator). The incubation under stirring continued for 90 min and the reaction was stopped by the addition of 5 ml 4 N sodium acetate buffer pH 5.5. The protein was separated from t,he low-molecular-weight substances by gel filtration on Sephadex 6-25 (coarse) equilibrated with 0.001 N HC1 and lyophilized.I n the studies with 14C-labelled compound I1 the incorporation of the latter into the protein was determined on the aliquots of samples separated from excess reagents by the gel filtration using a Mark1 (Nuclear Chicago) scintillation counter. The composition of the scintillation mixture was as follows: 4 g PPO, 2 g POPOP, 60g naphthalene, 200ml ethanol and 800 ml dioxane.The protease and peptidase activities were determined using hemoglobin

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Three MAb M2, B6, P1 (IgG1 type) against human urinary trypsin inhibitor (UTI), a glycoprotein with antiinflammatory properties, have been produced by hybridization of mouse myeloma cells P3o1 with spleen cells of immunized mice BALB/c. Competitive ELISA-examination of peroxidase conjugates of M2, B6, and P1 MAb in the presence of the trypsin binding domain shows the highest affinity of M2 antibody for this domain. On the basis of MAb M2 competitive ELISA of UTI concentration in urine is proposed. ELISA detectable changes in the UTI content of urine from patients with nephritis without renal failure can be considered as an early index of renal parenchyma damage.

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