The peptide-bond-specificity of bovine spleen cathepsin S in the cleavage of the oxidized insulin B-chain and peptide methylcoumarylamide substrates was investigated and the results are compared with those obtained with rat liver cathepsins L and B. Major cleavage sites in the oxidized insulin B-chain generated by cathepsin S are the bonds Glu13-Ala14, Leu17-Val18 and Phe23-Tyr26; minor cleavage sites are the bonds Asn3-Gln4, Ser9-His10 and Leu15-Tyr16. The bond-specificity of this proteinase is in part similar to the specificities of cathepsin L and cathepsin N. Larger differences are discernible in the reaction with synthetic peptide substrates. Cathepsin S prefers smaller neutral amino acid residues in the subsites S2 and S3, whereas cathepsin L efficiently hydrolyses substrates with bulky hydrophobic residues in the P2 and P3 positions. The results obtained from inhibitor studies differ somewhat from those based on substrates. Z-Phe-Ala-CH2F (where Z- represents benzyloxycarbonyl-) is a very potent time-dependent inhibitor for cathepsin S, and inhibits this proteinase 30 times more efficiently than it does cathepsin L and about 300 times better than it does cathepsin B. By contrast, the peptidylmethanes Z-Val-Phe-CH3 and Z-Phe-Lys(Z)-CH3 inhibit competitively both cathepsin S and cathepsin L in the micromolar range.

A B S T R A C TKey words: Rapeseed detoxification, seed flour, functional properties, foaming capacity, water absorption, cooking loss, amino acid composition. INTRODUCTIONThe increased interest in rapeseed as a source of edible oil and protein (Appelquist and Ohlson 1972;Bunting 1986) Few investigations of the functional properties of the resulting rapeseed flours or protein concentrates have been published and in these an acceptable or good functionality has been reported. However, one has to draw attention to the deteriorating effect of heat treatment on the functional properties of rapeseed protein products reported by Hermanson et a1 (1974) and Ohlson and Anjou (1979). A chemical or enzymic modification of such a protein concentrate was necessary to obtain a product with acceptable functionality (Hermanson et a1 1974). Moreover, heat treatment of intact rapeseeds or rapeseed meals not only causes aggregation and denaturation of proteins but also favours undesirable interactions between proteins and non-protein compounds.The present paper is concerned with a procedure for the preparation of detoxified and high-functional rapeseed flours which consists of the first stage of a lowtemperature soaking of intact seed under mild acidic or alkaline conditions. EXPERIMENTAL MaterialsThe rapeseed flours were prepared from a low-erucic acid variety 'Marinus' of Brassica napus (harvested 1986). Ammonium carbamate (Merck, Darmstadt, FRG), citric acid (Chemapol, Prague, CSSR) and Reinecke salt (Fluka AG, Buchs, Switzerland) were analytical reagent grade.Preparation of rapeseed flours (Fig 1) Ammonium carbarnate variant Rapeseed (1 kg) was soaked overnight with 1 litre of 50g litre-' ammonium carbamate solution at room temperature (pH 7-9). The procedure was repeated after draining the swollen seeds by suction. The seeds were then dried on filter paper at room temperature and finally kept for 2 h at 60°C in an oven. The dried seeds (900g) were crushed in a roll crusher. The dehulling was performed using air classification after screening the raw meal through a 2-mm sieve. Then the meal was defatted by diethyl ether extraction in a Soxhlet apparatus. The defatted material was finely ground and screened. The fraction with particle diameter < 160 pm was used for the final dehulling step. The latter consisted of flotation of the small hull fraction still remaining in the meal using hexane according to Sosulski and Zadernowski (1981); 160 g of a light creamcoloured flour was obtained (sample 1). Citric acid variantRapeseeds (1 kg) were soaked for 3 h with 1 litre l o g litre-' citric acid monohydrate and filtered under suction. The pH of the filtrate was 4.0. The procedure was repeated three times. The pH of the last filtrate was 3.5. After drying, defatting and dehulling as in the foregoing procedure, 150g of a light cream coloured flour with a particle diameter < 160 pm was obtained (sample 2). Alcohollammonia treatmentTen grams each of samples 1 and 2 above were stirred with 80 ml of a mixture of methanol/ammonia (85 vol m...

Rat liver cathepsin B was tested for its peptide-bond specificity against bradykinin and the oxidized insulin A-chain. Bradykinin was shown to be resistant to the action of cathepsin B. One possible reason for this resistance is the proline content of the peptide and the discrimination against proline residues at three or four subsites of cathepsin B. Oxidized insulin A-chain was degraded by a peptidyl dipeptidase activity. Three dipeptides were cleaved from the C-terminal part of the insulin A-chain after having been incubated for 2 h (molar ration E:S = 1:2800) and six dipeptides were released after a longer digestion (10 h, E:S = 1575). Cathepsin B; Peptidyl dipeptidase; Substrate specificity; Bradykinin; Oxidized insulin A-chain

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