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Two separation methods, ultrafiltration and 2-butanol extraction, have shown that a peptide is the major agent responsible for bitterness in peptic hemoglobin hydrolysates. It was easily purified from these complex mixtures by specific hydrophobic adsorption on Superose 12, a gel-filtration column, which could constitute an original and interesting method for bitterness detection. The bitter peptide which corresponded to VV-hemorphin 7, the fragment 32-40 of the beta chain of bovine hemoglobin, is first generated during proteolysis, then hydrolysed by pepsin. It exhibited a strong bitterness at 0.25 mM equivalent to 0.073 mM quinine sulfate or 21 mM caffeine.

A new series of N-alkylated uric acids (2,6,8-purinetrione) and 5,6-diaminouracils (5,6-diamino-2,4-pyrimidinedione) were synthesized, and their activities against free radicals were evaluated. Long-chain derivatives of both series exhibited a large inhibitory activity against oxygen radical induced lipid peroxidation in bovine heart mitochondria (IC50 lower than 1 microM), compared to the reference antioxidants trolox C or alpha-tocopherol. This activity appeared related to (i) the ability of these compounds to reduce the stable radical 1,1-diphenyl-2-picrylhydrazyl and (ii) their lipophilicity estimated by log P determination. In order to study the scavenging mechanisms of diaminouracils and urate derivatives against lipid radicals, they were also tested against the azo-initiated peroxidation of either methyl linoleate in organic solvents or a liposomal suspension of dilinoleoylphosphatidylcholine. Urate derivatives reacted moderately with lipid radicals and were slowly consumed, significantly affecting the propagation of the peroxidation. Diaminouracils strongly reduced the propagation rate. They were quickly consumed and were able to deactivate about 1 mol of lipid radical per mole of compound in organic solvent. Dodecyl urates and decyl- and dodecyldiaminouracils were chosen for further in vitro investigation and in vivo evaluation.

A new series of metal ligands containing the 1,3-dimethyl-6-amino-5- nitrosouracil moiety has been synthesized and they have been studied as potential inhibitors of iron-dependent lipid peroxidation. For this purpose, these new derivatives have been tested in the Fenton induced deoxyribose degradation assay, which allows a quantitative measurement of their inhibitory effect towards hydroxyl radical generation. When iron(II) is complexed by these ligands, a strong inhibition of deoxyribose degradation is observed, especially in the case of tris-[2-(1,3-dimethyl-5-nitrosouracil-6-yl)aminoethyl] amine (5). This inhibitory effect is clearly related to a specific complexation of iron(II) and is not due to the direct scavenging of hydroxyl radical by the ligand. Inhibition of the iron mediated Fenton reaction presumably results from inactivation of the reactivity of the metal center towards hydrogen peroxide. These derivatives, as well as long alkyl chain substituted nitrosouracils were evaluated in the protection of biological membranes against lipid peroxidation (induced by iron(II)/dihydroxyfumaric acid and determined with the 2-thiobarbituric acid test). Ligand 5 inhibited lipid peroxidation at a rate similar to Desferal (desferrioxamine B) and slightly higher than bathophenanthroline sulphonate (BPS), which are respectively good iron(III) and iron(II) chelators. When covalently bound with a long alkyl chain, the increase of lipophilic character of the ligand allows its location near the mitochondrial membrane, where lipid peroxidation occurs. Lower concentrations (IC50 = 4 microM) are then necessary to inhibit lipid peroxidation. This IC50 concentration should be compared to those obtained for Trolox (IC50 = 3 microM) or the 21-aminosteroid U74500A (IC50 = 1 microM) described previously.

Histidine residues in horseradish peroxidase (HRP) were modified chemically with diethyl pyrocarbonate, 4,omega-dibromoacetophenone or diallylpyrocarbonate. Histidines were chosen as His-170, the fifth ligand of the heme iron atom, forms part of the active site of this enzyme. Good yields of hemoprotein were obtained in all cases. Analysis by HPLC of peptides obtained after tryptic digestion showed that His-170 of HRP was in fact modified. The specific activity remained satisfactory after chemical modification of the histidine residues, and so the active site of HRP can thus be altered without a dramatic loss of hemoprotein or peroxidase activity. This may open routes to the preparation of novel biocatalysts.

An immobilized enzyme electrode for continuous creatinine determination in blood serum is described. The enzymes creatinine amidohydrolase, creatine amidinohydrolase, and sarcosine oxidase are coimmobilized to the surface of the polypropylene membrane of a Clark-type electrode responsive to oxygen. The immobilized enzymes catalyze the decomposition of creatinine with the consumption of oxygen and thus permit the creatinine measurement. The whole assay takes less than 1 min. Effects of pH and temperature on electrode response are also described. The proposed technique offers a rapid, simple, and inexpensive means to determine creatinine in blood serum within the normal and abnormal ranges. The repeatability of the creatine determination in serum is 2.5% (relative standard deviation), and the detection limit is 3 x 10(-6) mol L-1. The results obtained by this method were compared to those obtained with the Technicon AutoAnalyzer SMAC system based on the Jaffé reaction; the correlation factor between the two methods was found to be r = 0.9997.

The effect of manganese on lignin peroxidase activity was studied. The enzyme was produced with a new process using an air-lift-type reactor. The experiments were performed with veratryl alcohol and a dimeric lignin model compound. It was shown that when Mn(II).lactate complex was present the amount of veratraldehyde formed and the uptake of oxygen were significantly enhanced during the aerobic oxidation of veratryl alcohol. A similar effect can be obtained with superoxide dismutase. These results strongly suggest that the superoxide anion can occur during the reaction; its scavenging by Mn(II) or superoxide dismutase generates H2O2. In contrast, no evidence for the formation of superoxide anion was found during oxidation of the lignin model, compound veratrylglycerol-beta-guaiacyl ether.