Proceedings of the Biochemical Society

Abstract

For many animal products, reaction with fluoro-2:4dinitrobenzene (FDNB), followed by acid-hydrolysis and ether-washing left solutions in which only E-DNP-lysine appeared to give significant colour (Carpenter & Ellinger, 1955). This measure of lysine related closely to the products' 'gross protein values' (G.P.V.) for chicks in assays where conditions make lysine the usual limiting factor (Carpenter, Ellinger, Munro & Rolfe, 1957). Vegetable materials gave interfering colours in this procedure. A modification uses the reaction of methoxycarbonyl chloride (MC-Cl) with a-amino-groups (Chibnall & Spahr, 1957); with E-DNP-lysine one obtains the ethersoluble (Bailey, 1957): DNP-NH* (CH2)4* CH(COOH) * NH-CO OCH3. Ground material is shaken with FDNB as before. We now hydrolyse with 24 ml. 8-1 N-HCI, reflux 16 hr. and make the filtrate to 100 ml. with water. 0-3-1-0 ml. is added to a glass-stoppered tube, extracted twice with 5 ml. di-ethyl ether, and dissolved ether removed by warming. The residue is made alkaline with 2 N-NaOH (after titration of a dummy aliquot plus phenolphthalein) and shaken with 2ml. of a buffer (8 % w/v NaHCO3 and Na2CO3 mixed to give pH 8-5). 0-05 ml. MC-Cl is added with shaking and, after 10 min., 0-75 ml. conc. HCI. Two 5 ml. portions of di-ethyl ether are then shaken in the tube, recovered and evaporated. The residue is redissolved in 10 ml. 1 N-HCI with warming, and read in an absorptiometer (601 filter: 435 mjL). The extinction is compared with those for E-DNP-lysine aliquots carried through the procedure, and the lysine equivalent calculated. Nine samples of oilseed , cereal and grass products (Carpenter, Duckworth, Ellinger & Shrimpton, 1955) and eight samples of leaf protein concentrates processed in different ways (Cowlishaw, Eyles, Raymond & Tilley, 1956) gave values from 1-9 to 4.9 g. lysine/16 g. N, which showed a highly significant correlation (r = 0-84) with their G.P.V. figures. The procedure may therefore prove useful in grading vegetable products. Unfortunately, carbohydrates can increase the loss of DNP-lysine during hydrolysis. Also, chromatography with mixed organic solvents on buffered paper (Blackburn & Lowther, 1951) reveals an interfering band in some cases, though on elution this has shown little extinction at 435 m,u. We thank Dr K. Bailey and Dr F. Sanger for advice and chemicals, the Grassland Research Institute for test samples, and du Pont and Co. for a grant (D. B.).