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The levels of amino acids in broccoli stems and florets before and after various blanching times (in the case of freezing) and after bottling have been studied to elucidate to what extent nutrient quality is affected by industrial processing. The following amino acids (mg kg^-1 fresh weight) were identified by ion exchange chromatography in raw broccoli florets: glutamine (1338), proline (732), asparagine (578), valine (310), arginine (296), isoleucine (204), threonine (169), leucine (166), phenylalanine (159), aspartic acid (140), lysine (127), alanine (122), tyrosine (105), S-methylcysteine (96), histidine (89), ornithine (59), glutamic acid (44), γ-aminobutyric acid (31), glycine (11) and serine (0.2). Raw stems contained the same amino acids but at lower levels (p < 0.05). The levels of all these amino acids fell during both industrial processes studied (bottling and freezing after blanching for 60, 90, 120 and 150 s), particularly in the frozen samples (losses of 50-70% in the florets and 20-50% in the stems). In summary, losses of broccoli amino acids were lower if blanching times were kept short. The optimal blanching time at 94 °C for florets and stems intended for freezing was 90 s, and this did not result in any great loss of nutritional value related to amino acid content. Bottled florets had greater nutritional value than those frozen after being exposed to the longest blanching times (120 and 150 s).
© 2001 Society of Chemical Industry

The antioxidant and pro-oxidant activities of broccoli amino acids were compared with those of common food additives. In decreasing order, the data showed that Asp, SMC, GABA, Glu, Gln, Pro, Phe, Leu, Lys, Arg, Asn, Val, Ile, His, Ser, Gly, Orn and Ala, when dissolved in water at concentrations of 0.5 and 0.05 mM, partially inhibited damage to deoxyribose in the presence of ferric-EDTA and H₂O₂. In contrast, Tyr and Thr acted as pro-oxidants in this system. The amino acids present in broccoli had no hydrogen peroxide-scavenging effect. When dissolved in water, methanol or ethanol, SMC, Glu, Thr, Gln, Ser, GABA, Pro, Ala, Ile, Phe, Asp, Orn and Tyr inhibited lipid peroxidation. However, Asn, Val, Arg, Leu, Lys, His and Gly were not effective in decreasing peroxidation at concentrations of 0.5 and 0.05 mM. Asp > SMC > Ala > Phe > Hys > Orn > Gln = Ser > Lys > Leu = GABA = Gly > Tyr > Arg = Thr > Val > Asn > Pro > Ile > Glu (p < 0.025) showed scavenging activity towards hypochlorous acid, protecting α₁-antiproteinase against inactivation. In this paper it has been established that some amino acids premixed with propyl gallate increase its hypochlorous acid-scavenging capacity, while other amino acids have an additive effect with propyl gallate, permitting smaller quantities of propyl gallate to be used as food additives in some products which contain these amino acids.
© 2001 Society of Chemical Industry

Acid phosphatase assay with alpha-naphthyl phosphate as substrate and the use of diazonium salt (Fast Red TR) for chromophore formation was kinetically analysed as a system of two chemical reactions coupled to an enzymic reaction. This system follows a mechanism defined as enzymic-chemical-chemical (EzCC). The accumulation of chromophore with reaction time presented a marked lag period, which was only dependent on the rate constants of the chemical reactions and was independent of the enzymic step. The specific rate constants of each chemical step were determined in 3.8-5.0 pH and 10-35 degrees C temperature ranges. Thermodynamic parameters of the chemical steps were also obtained. Measurement of acid phosphatase activity can be carried out in the pH range 3.8-5.0 (4.8 was optimal pH) without the need to eliminate the lag period.

Ca2+ + Mg2+-dependent ATPase from sarcoplasmic reticulum was inhibited by preincubation with vanadate. When the inhibited enzyme was preincubated in the presence of vanadate and assayed in its absence, a slow reactivation process was observed. This slow, hysteretic, process was exploited to study the influence of Ca2+ and ATP on the dissociation of vanadate. Ca2+ alone slowly displaced vanadate from the inhibited enzyme, and a rate constant of 0.1 min-1, at 25 degrees C, was calculated for this re-activation process. However, ATP re-activated with an apparent constant that hyperbolically depended on ATP concentration, and from it a rate constant for vanadate dissociation induced by ATP of 0.5 min-1 was calculated. It is deduced from the kinetic studies that ATP binds to the enzyme-vanadate complex, forming a ternary complex, with a dissociation constant of 4 microM, and that this binding accelerates vanadate dissociation. Binding experiments with [14C]ATP showed that ATP binds to the enzyme-vanadate complex with a dissociation constant of 12 microM, i.e. the affinities calculated with the isotope technique and the kinetic procedure are of the same order of magnitude.

Having oxidized 3,4-dihydroxyphenylalanine (dopa) with sodium periodate or mushroom tyrosinase in a pH range from 3.5 to 6.0, it has been possible to detect spectrophotometrically 4-(2-carboxy-2-aminoethyl)-1,2-benzoquinone with the amino group protonated (o-dopaquinone-H+), a postulated intermediate in the melanogenesis pathway. When the pH value was greater than 4, the final product obtained was 2-carboxy-2,3-dihydroindole-5,6-quinone (dopachrome); however, for pH values lower than 4, two different products were identified by means of cyclic voltammetry: 5-(2-carboxy-2-aminoethyl)-2-hydroxy-1,4-benzoquinone and dopachrome. These products appeared when oxidation was achieved with the enzyme as well as with periodate. This suggests that two chemical pathways can arise from alpha-dopaquinone-H+, whose relative importance is determined by the pH. The steps of these pathways would be dopa leads to o-dopaquinone-H+ leads to o-dopaquinone leads to leukodopachrome leads to dopachrome, for the first one, and dopa leads to o-dopaquinone-H+ leads to 2,4,5-trihydroxyphenylalanine leads to 5-(2-carboxy-2-aminoethyl)-2-hydroxy-1,4-benzoquinone very slowly leads to intermediate compound leads to dopachrome, for the second one. The stoichiometry for the conversion of dopaquinone-H+ into dopachrome for pH values greater than 4 followed equation of 2 o-dopaquinone-H+ leads to dopa + dopachrome. No participation of oxygen was detected in the conversion of leukodopachrome (2,3-dihydro-5,6-dihydroxyindole-2-carboxylate) into dopachrome.

Tyrosinase usually catalyses the conversion of monophenols into o-diphenols and the oxidation of diphenols to the corresponding o-quinones. Sugumaran [(1986) Biochemistry 25, 4489-4492] has previously proposed an unusual oxidative decarboxylation of 3,4-dihydroxymandelate catalysed by tyrosinase. Our determination of the intermediates involved in the reaction demonstrated that 3,4-dihydroxybenzaldehyde is not the first intermediate appearing in the medium during the enzymic reaction. Re-examination of this new activity of tyrosinase has demonstrated that the product of the enzyme action is the o-quinone, which, owing to its instability, evolves to the final product, 3,4-dihydroxybenzaldehyde, by a chemical reaction of oxidative decarboxylation.