We have synthesized two sets of noncleavable peptide-inhibitor libraries to map the S and S' subsites of human heart chymase. Human heart chymase is a chymotrypsin-like enzyme that converts angiotensin I to angiotensin II. The first library consists of peptides with 3-fluorobenzylpyruvamides in the P1 position. (Amino acid residues of substrates numbered P1, P2, etc., are toward the N-terminal direction, and P;, P2, etc., are toward the C-terminal direction from the scissile bond.) The Pi and P' positions were varied to contain each one of the 20 naturally occurring amino acids and P3 was kept constant as an arginine. The second library consists of peptides with phenylalanine keto-amides at P1, glycine in PN, and benzyloxycarbonyl (Z)-isoleucine in P4. The P2 and P3 positions were varied to contain each ofthe naturally occurring amino acids, except for cysteine and methionine. The peptides of both libraries are attached to a solid support (pins). The peptides are evaluated by immersing the pins in a solution of the target enzyme and evaluating the amount of enzyme absorbed. The pins with the best inhibitors will absorb most enzyme. The libraries select the best and worst inhibitors within each group of peptides and provide an approximate ranking of the remaining peptides according to Ki. Through this library, we determined that Z-Ile-Glu-Pro-Phe-CO2Me and (F)-Phe-CO-Glu-Asp-ArgOMe should be the best inhibitors of chymase in this collection of peptide inhibitors. We synthesized the peptides and found K1 values were 1 nM and 1 ,uM, respectively. The corresponding K1 values for chymotrypsin were 10 nM and 100 ,uM. The use of libraries of inhibitors has advantages over the classical method of synthesis of potential inhibitors in solution: the libraries are reusable, the same libraries can be used with a variety of different serine proteases, and the method allows the screening of hundreds of compounds in short periods of time.Inhibitors of proteolytic enzymes generally have the following

A gas chromatography with flame ionization detection method (GC-FID) with direct injection, using a capillary column, was validated to determine ethanol, acetaldehyde, methanol, and acetone in different human matrices, such as whole blood, vitreous humour, and urine, with clinical and forensic interest. This method was also employed to quantify these compounds in cell culture medium, thus being useful in basic research. A good peak resolution was achieved, with linear correlation between concentration and peak areas for all the compounds in all the matrices. The inter- and intra-day precisions of the method were always under 15% and 10%, respectively. The accuracy of the method, calculated as the percentage of the target concentration, was within the acceptable limits. The obtained limits of detection were below 0.85 mg/L for acetaldehyde and below 0.75 mg/L for the other considered compounds. The small injection volume and the high split ratios applied, allied to the high performance of the GC column, resulted in very good peak resolution and high sensitivities. This method is easy to perform, making it suitable for the routine of clinical biochemistry and forensic laboratories.

Oxidative stress induced by catecholamines is a well recognized toxic event. This effect has been extensively observed in the heart, where high levels of catecholamines cause enzyme inhibition, lipid peroxidation, energy depletion and myocardial necrosis. Catecholamines can be converted into o-quinones and undergo cyclization into aminochromes. This process can occur enzymatically or through autoxidation and involves the formation of free radicals. Aminochromes are highly reactive molecules that can cause oxidation of protein sulfhydryl groups and deamination catalysis, among other deleterious effects; in addition, inhibition of some enzymes has been also reported. We have studied the effects of isoproterenol oxidation products (IOP) on glutathione reductase (GR) activity in vitro. Isoproterenol (ISO) autoxidation was conducted at 37 degrees C in the dark, for 4 h at pH 7.0 and this process was monitored by UV spectrophotometry at both 340 and 490 nm. Addition of the autoxidized solution to GR in the presence of oxidized glutathione (GSSG) and NADPH showed that IOP inhibits GR in a competitive mode and that this effect increases during the 4 h incubation period. This inhibitory effect of IOP was partially prevented by the addition of reduced glutathione (GSH), L-cysteine and ascorbic acid to the reaction mixtures.

An electrothermal atomic absorption spectrometric method was developed for the determination of total and hexavalent chromium in animal feeds. For measuring total chromium the samples were digested in a mixture of three acids (HN0,-HCI-HF). Chromium in its hexavalent state was dissolved in 0.01 mol I-' NaOH solution. A mixture of Pd+Mg was used as a chemical modifier. Extensive validation of the proposed method was carried out both by the standard additions method and by analysis of National Institute of Standards and Technology Standard Reference Material 1548 Total Diet. The detection limits were 0.53 and 0.42 pg I-' for total and hexavalent chromium, respectively. Measurements can be made over a linear range between 0.53 and 50 and 0.42 and 50 pg I-' for total and hexavalent chromium, respectively. The relative standard deviations were 4.5 and 7.0% for total and hexavalent chromium, respectively, hence the proposed method is satisfactory for routine analyses. In 70 samples that were analysed the mean levels found were 1.93 pg g-' (0.20-6.87 pg g-') and 230 ng g-' (10.0-780 ng g-I), for total chromium and chromium(vi), respectively. The large variations in the levels of metal found in the analysed feeds shows diverse contamination of the raw materials used in the preparation of the samples.