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beta-Lactoglobulin is a whey protein that affects milk composition and product functionality and which can be present in up to eight genetic variant forms. A free zone capillary electrophoresis method has been developed to separate and identify the beta-lactoglobulin A, B and C variants. Three buffer systems [borate, 2-(N-morpholino)-ethanesulphonic acid (MES) and bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane (Bistris)] were examined over a range of pH values and with the addition of the separation buffer modifiers Tween 20 and/or ethanolamine. The most successful combination of these was 50 mM MES at pH 8.0 with the addition of 0.1% Tween 20 which clearly resolved the three variants from both each other and from the other whey proteins even though the MES buffer was acting well outside its pKa range (pH 5.3-7.3). The retention times and identification of the individual variants were verified by spiking with commercially purified beta-lactoglobulin A and B proteins and a beta-lactoglobulin AC whey. The method was then used to phenotype beta-lactoglobulin in a sample population of New Zealand Jersey cows.

Sheep liver cytoplasmic aldehyde dehydrogenase was labelled by reaction with the substrate p-nitrophenyl di[14C]methylcarbamate. After tryptic digestion and peptide fractionation the labelled residue was identified as Cys-302. This is the first unequivocal identification of the essential enzymic nucleophile in the esterase activity of aldehyde dehydrogenase. By implication, Cys-302 is probably also the residue that is acylated by aldehyde substrates and the first residue that is modified by disulfiram.

The kcat value for the oxidation of propionaldehyde by sheep liver cytosolic aldehyde dehydrogenase increased 3-fold, from 0.16 s-1 at pH 7.6 to 0.49 s-1 at pH 5.2, in parallel with the increase in the rate of displacement of NADH from binary enzyme.NADH complexes. A burst in nucleotide fluorescence was observed at all pH values consistent with the rate of isomerization of binary enzyme.NADH complexes constituting the rate-limiting step in the steady state. No substrate activation by propionaldehyde was observed at pH 5.2, but the enzyme exhibited dissociation/association behavior. The inactive dissociated form of the enzyme was favored by low enzyme concentration, low pH, and low ionic strength. Propionaldehyde protected the enzyme against dissociation.

Pyrophosphate ions activate the steady-state rate of oxidation of propionaldehyde by sheep liver cytosolic aldehyde dehydrogenase at alkaline pH values. The steps in the mechanism governing the release of NADH from terminal enzyme. NADH complexes have been shown to be rate-limiting at pH 7.6 [MacGibbon, Buckley & Blackwell (1977) Biochem J. 165, 455-462]. These steps are shown to be also rate-limiting at more alkaline pH values, and it is through an acceleration of these steps that pyrophosphate ions exert their activation effect.

p-Chloromercuribenzoate (PCMB) at stoichiometric levels reacts with a thiol group of the binary NAD+ complex of sheep liver cytoplasmic aldehyde dehydrogenase (E.NAD+) faster than with the corresponding thiol group of either the free enzyme or the binary enzyme. NADH complexes. High concentrations of propionaldehyde have a protective effect against modification of the enzyme with PCMB in steady-state assays. This protection arises from a reduction in the concentration of the E.NAD+ binary complex rather than competition for a common binding site. PCMB has three major effects on aldehyde dehydrogenase. First, rapid reaction with a high-affinity thiol group in the E.NAD+ binary complex causes activation of the steady-state rate. The activation results from an increase in the rate of NADH release from the enzyme. This modification simultaneously protects against dilution-induced dissociation of enzyme tetramers. Second, premodification of the high-affinity thiol group leads to inhibition of the steady-state rate at high propionaldehyde concentrations, because of the increased affinity of the free enzyme for propionaldehyde with the resultant formation of an enzyme-aldehyde dead-end complex. Third, when higher ratios of PCMB to enzyme (> 3:1) are used, one or more other thiol groups are also modified, causing enzyme dissociation and subsequent inactivation. Since modification of the high-affinity thiol by PCMB causes activation, clearly it cannot be the active site acylation center involved in propionaldehyde oxidation. The different amplitudes of the proton burst at high and low propionaldehyde concentrations for the PCMB modified enzyme provide support for a second binding site for propionaldehyde on the enzyme.

Although studies on the effect of pH on aldehyde dehydrogenase have been carried out in a number of laboratories, the results of these studies do not give a complete picture of the mechanism of the aldehyde dehydrogenase catalyzed oxidation of propionaldehyde over an extended pH range. The enzyme catalyzed oxidation reaction at pH 7.0 and 7.6 is generally agreed to occur by the following ordered mechanism: $$E \rightleftharpoons E.NA{D^ + } \rightleftharpoons E.NA{D^ + }.ALD \rightleftharpoons E.NADH.acyl \to *E.NADH \rightleftharpoons E.NADH \rightleftharpoons E$$ Scheme I At least at low propionaldehyde concentrations, the rate of the reaction is controlled by the slow conformational change which occurs during the release of NADH from the enzyme (Blackwell et al., 1987; Dickinson, 1985).

The release of NADH from the enzyme·NADH complexes was rate limiting at 37°, for the oxidation of propionaldehyde by sheep liver cytosolic aldehyde dehydrogenase. Marked substrate activation was observed at this temperature as was activation by p -(chloromercuri)benzoate. Activation of enzymic activity may be of importance in vivo .

1. The pig enzyme was purified to homogeneity and was found to be a tetramer of apparently identical subunits. 2. The pig enzyme was found to contain 1 mol NADH/mol enzyme which is tightly bound, which is not directly involved in catalysis and which so far has not been removed from the enzyme so as to produce an active apoenzyme. 3. The pig enzyme seems to contain only one functioning active site/tetramer. 4. The pig and sheep enzymes are compared in respect of NADH binding, substrate specificity, immunological response and surface charge.