Simplified determination of lorazepam and oxazepam in biological fluids by gas chromatography—mass spectrometry

Lorazepam and oxazepam in plasma and urine were measued by gas chromatography—mass spectrometry. Oxazepam was used as an internal standard in the assay of lorazepam and vice versa. After removal of interfering substances with n-hexane, the drugs were extracted with benzene and converted to N₁,O₃-bistrimethylsilyl derivatives. Glucuronide forms of the drugs were extracted after hydrolysis with β-glucuronidase. A common fragment ion at m/e 429 was used to monitor the two drugs. The sensitivity was 2 ng/ml for both drugs, which was sufficient to determine plasma and urine concentrations after therapeutic doses to humans.     

Test reactions for a stopped-flow apparatus: Reduction of 2,6-dichlorophenolindophenol and potassium ferricyanide by l-ascorbic acid

A reaction system suitable for testing the function of a stopped-flow apparatus of high performance was searched for. The reduction of 2,6-dichlorophenol-indophenol by l-ascorbic acid at pH 2.0 was recommended as the most practical test reaction. In due course of the study the reaction mechanism of the reduction was discussed.     

An unusual fluorescence spectrum of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor, a dimeric protein proteinase inhibitor isolated in crystalline form by Murae et al. in 1972, contains three tyrosine and one tryptophan residues per monomer unit and has unusual fluorescence properties. When excited at 280 nm, it shows a characteristic fluorescence spectrum having a peak at 307 nm and a shoulder near 340 nm, a feature which has been recognized only for a very few cases in proteins containing both tryosine and tryptophan residues. When excited at 295 nm, at which tryrosine scarcely absorbs, the inhibitor shows an emission spectrum with a peak at 340 nm characteristic of a tryptophan residue. The emission with a peak at 307 nm is considered to arise from the tryrosine residues. The tryptophan quantum yield of Streptomyces subtilisin inhibitor excited at 295 nm is very small, indicating that the tryptophan florescence is strongly quenched in the native state of the inhibitor. Below pH 4 the peak of the fluorescence spectrum of the inhibitor excited at 280 nm shifts toward 340-350 nm with a concomitant increase in the quantum yield. The structural change induced by low pH seems to release the tryptophan fluorescence from the quenching.     

Kinetic study of isomerization of ferricytochrome c at alkaline pH

Kinetic studies of the isomerization reaction of horse heart ferricytochrome c between pH 8.5 and pH 12.1 have been carried out by using stopped-flow and rapid scanning stopped-flow techniques. Below pH 10, our results were in good agreement with the scheme proposed earlier (Davis, L. A., Schejter, A. and Hess, G. P. (1974) J. Biol. Chem. 249, 2624–2632). Above pH 10, another faster first-order process was observed, which suggested the existence of a transient species in the isomerization reaction between the species with and without a 695 nm band. The probable scheme of the isomerization reaction is considered to be

where H denotes a proton, the colored forms are the species predominant at neutral pH with a 695 nm band and the noncolored forms are the species without a 695 nm band. The transient species has a small 695 nm absorbance which suggests that the sixth ligand is still Met-80, although the protein conformation might be different from that at neutral pH.     

Temperature-jump studies on benzeneboronic acid-serine protease interactions. Subtilisin and alpha-chymotrypsin.

The interactions of benzeneboronic acid (BBA) as a transition state analog with subtilisin (EC 3.4.21.4) and with alpha-chymotrypsin (EC 3.4.21.1) were investigated kinetically by the temperature-jump method using pH indicators. For both enzymes, the concentration dependence of the relaxation time was consistent with a two-step mechanism involving a fast bimolecular association followed by a slow, unimolecular process. The possibility of a trigonal-tetrahedral interconversion of BBA at the active site of the enzyme is discussed.     

The pH jump study of enzyme proteins. I. Liquefying alpha-amylase from Bacillus subtilis.

Rapid conformational changes due to pH jump were studied kinetically at 25 degrees mainly by the stopped-flow method using liquefying alpha-amylase from Bacillus subtilis [EC 3.2.1-.1, liquefying]. First, the conformational change due to a pH jump produced by mixing with alkali was monitored as a function of time at 245 nm through the ionization of phenolic hydroxyl groups of tyrosine residues which were originally buried and finally become exposed due to the pH jump. Three distinct phases of conformational change were clearly recognized by this method by varying the final pH values. Each phase involved the exposure of an essentially definite number of tyrosine residues, whose rate constant was crucially dependent on pH. Second, these phases of conformational change were subjected to examination in terms of the optical rotation change at 411 nm and the reversibility upon reverse pH jump with respect to conformational reconstitution, as observed through the protonation ofphenolic hydroxyl groups of ionized tyrosine residues and the enzyme activity. The first phase, which occurs above pH 12.5, involves no change in the optical rotation and is reversible as observed by the above two monitoring methods. In contrast, the other two phases, which are observed above pH 12.7, are accompanied by an optical rotation change and no appreciable reversibility was detected by these methods.     

Genetically engineered rice containing larger amounts of nicotianamine to enhance the antihypertensive effect

Nicotianamine (NA), a metal chelator ubiquitous in higher plants, serves as an antihypertensive substance in humans. To engineer a novel antihypertensive rice that contains larger amounts of NA, the barley NA synthase gene, HvNAS1 , was introduced into rice via Agrobacterium -mediated transformation. The introduced HvNAS1 was driven by pGluB-1 , which induces strong gene expression in the endosperm of rice seeds. The NA content in transgenic rice seeds was up to fourfold greater than that in non-transgenic rice seeds. The Cre/ loxP DNA excision (CLX) system was used to remove the selectable marker gene for antibiotic resistance. Furthermore, the transgenic rice was crossed with a cleistogamous mutant to prevent gene transfer via pollen dispersal. These two modifications may minimize public concern with regard to the use of this transgenic rice.