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Polyphenol oxidase extracted from oil bean (Pentaclethra macrophylla) seeds was purified 165-fold over the crude enzyme extract. The apparent molecular weight of the enzyme by gel filtration was 110.8 kf9.0 k while SDS-PAGE indicated a single species of molecular weight 28.0 k. A copper content of 1.9 mg g-' corresponds to one copper atom for each of the four subunits. The purified enzyme oxidised pyrogallol, catechol, 4-methylcatechol and Ldihydroxyphenylalanine but had low activity towards tyrosine. p-Cresol, caffeic acid and cholorogenic acid were not oxidised. Thio-compounds were strong inhibitors of the enzyme. The phenolic compounds tyrosine, resorcinol and orcinol inhibited catechol oxidation but became oxidised in the process.Key words : Pentaclethra macrophylla, oil bean seeds, polyphenol oxidase, purification, properties.Polyphenol oxidase (PPO) (o-diphenol : 0, oxidoreductase, EC 1.10.3.2) is a copper-containing enzyme which catalyses the o-hydroxylation of monophenols to o-diphenols and the oxidation of o-diphenols to 0-quinones. The enzyme can be found in a variety of species and, though it contributes to the discolouration of plant materials on injury, its functions in higher plants remain unclear (Vaughn and Duke 1984). Work on higher plant PPO is handicapped by the apparent difficulties in isolation and purification of the enzyme due to protein-quinone interactions and binding of enzyme to particles during grinding and fractionation (Mayer and Hare1 1979). Recent attempts to use affinity and hydrophobic chromatography on mushroom tyrosinase led to the discolouration of the matrix due to oxidation of coupled ligands (Ingebrigsten and Flurkey 1988). Apart from problems of isolation and purification, there is controversy over the number and type of polypeptide chains in higher plant PPO (Mayer 1987).The seeds of P macrophylla are eaten as a form of salad. The colour ranges from green to brown due to * Present address Department of Biochemistry, University of Port-Harcourt, Port-Harcourt, Nigeria. browning reaction. As these reactions cause loss of nutritive value of food (Jood et af 1987), it was decided to purify and characterise the enzyme from these seeds in order to control the browning. Table 1 shows the purification profile of the enzyme. Attempts to isolate the enzyme by ion-exchange chromatography from dialysed ammonium sulphate precipitates using a long column of DEAE-cellulose (30.0 cm x 2.0 cm id) and gradient elution were unsuccessful. However, stepwise elution of the buffer enzyme extract on a squat column (9.5 cm x 5.0 cm id) gave the enzyme in high purity.The enzyme was purified 165-fold with 20 % recovery of initial enzyme activity. The purified enzyme and bovine serum albumin (BSA) were separately electrophoresed in 75 g kg-' polyacrylamide gels in tris-glycine buffer, pH 8.3, and located by staining. The purified enzyme revealed a single band of mobility of 0.92 relative to BSA.Gel filtration of the purified enzyme according to Andrews (1 964) was with Sephadex G-200-120 c...

Thiocyanate levels were determined in serum and urine samples obtained from a human population sample of healthy non-smoking volunteers (aged between 14 and 30 years) of both sexes known to eat gari-based meals at least once a day. The samples were collected before and 3-4 hours after a gari- or rice-based meal. The values obtained before the test meals showed a wide variation, ranging between 39.20 +/- 1.95 to 160.95 +/- 8.06 mumol/l of serum, and 81.92 +/- 9.78 to 294.01 +/- 14.70 mumol/l of urine. For each volunteer, the serum and urine thiocyanate were affected by the test meals. Average increases of 18 and 20% were observed for serum and urine thiocyanate, respectively, following a gari-based meal. A rice-based meal produced, on the average, 10% decrease in both serum and urine thiocyanate. No significant effect of sex or age on the thiocyanate levels was observed. The gari samples used in the study, as well as random samples from the locality of study, had no detectable thiocyanate but contained between 0.013 and 0.015 mg cyanide per kg of gari. These findings indicate that conversion to thiocyanate is a significant pathway in the metabolism of HCN and contributes significantly to thiocyanate found in body fluids and tissues of man. In addition, support is provided for the possible involvement of the sulphur-transferases in the process of cyanide detoxication.

1. A purification procedure for the dimeric arginine kinase of the sea cucumber Holothuria forskali is described. 2. The enzyme has a mean molecular weight of 77250 and is composed of two equal, dissociable subunits. 3. It also shows co-operativity between substrate binding at one catalytic site to a much greater extent than the nomomeric lobster arginine kinase for which such co-operativity could not be detected unambiguously. The constants for substrate binding are reported assuming that the enzyme follows rapid-equilibrium random kinetics. From a comparison with other species, the development of co-operativity between the nucleotide- and guanidine-binding sites on one subunit is suggested to have occurred more than once in the evolution of the phosphagen kinases and is not dependent on subunit aggregation. 4. Both enzymes show similar pH profiles for thermal inactivation at 22 degrees C and have very similar stabilities. Above 40 degrees C the dimeric enzyme is much more stable than the monomer. Rate constants for heat inactivation and Arrhenius activation energies are reported. 5. The dimeric enzyme is also more stable to urea inactivation. Substrates and argininic acid all improve the stability of both enzymes. The effects of individual substrates are more distincitive with the dimeric enzymes and increase its stability to an extent that makes it about as stable as dogfish creatine kinase. In the physiological range dimerization does not seem to confer any particular advantage with respect to stability over the monomer form.

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1. The nature of arginine binding to lobster arginine kinase and the extent of its possible involvement with the ;essential' thiol group of the enzyme has been investigated with some inhibitory analogues of arginine. 2. Most of the analogues inhibit competitively, although mixed inhibition may occur if the alpha-carboxy group or alpha-amino group is absent. 3. The K(i) values indicate that strength of binding depends on the length of the carbon chain (l-isoleucine>l-valine>l- alpha-aminobutyrate>l-alanine) and the integrity of the substituents on the alpha-carbon atom (l-arginine>agmatine and l-ornithine>putrescine). The guanidino group probably contributes little to substrate binding, but a positive charge near the delta-nitrogen atom appears to be important (l-ornithine>l -citrulline>l-alpha-aminobutyrate). A cyclic analogue, 2-carboxymethyl-3-oxo-2,3,5,6,7,8-hexahydro-1H-imidazo [1,2-a][1,3]diazepine-8-carboxylic acid, has a low K(i) value similar to that of an equivalent straight-chain form, suggesting that arginine probably binds in a folded configuration. 4. The aliphatic l-amino acids give enzyme difference spectra similar to that with l-arginine and the integrity of the alpha-carboxy and alpha-amino groups appears to be a minimal but not sufficient requirement for this, as l-ornithine gives an atypical difference spectrum. A difference spectrum is interpreted as indicating an enzyme conformational change. No difference spectrum was observed with methylguanidine. 5. The ability of aliphatic alpha-l-amino acids to protect against inhibition by 5,5'-dithiobis-(2-nitrobenzoic acid) is proportional to the number of atoms in the carbon chain and inversely proportional to K(i). Ornithine gives greater protection than citrulline; analogues lacking the alpha-amino groups also protect. Agmatine, lacking the alpha-carboxy group, did not protect. 6. It is concluded that it is unlikely that the ;essential' thiol group in the enzyme interacts with any part of the arginine molecule during catalysis except, possibly, the alpha-carboxyl group.

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