Masatake Iwasaki

Chemiluminescence determination of guanine and its nucleosides and nucleotides using phenylglyoxal

Abstract A novel chemiluminescence method is described for the determination of guanine and its nucleosides and nucleotides. The method is based on the fluorescence reaction of the substances with phenylglyoxal in a phosphate buffer (pH 6.0) at 37°C for 20 min, followed by a chemiluminescence reaction with N,N -dimethylformamide in a weakly alkaline medium. The established conditions of the chemiluminescence reaction do not permit any development of chemiluminescence from other nucleic acid bases such as adenine, cytosine, uracil and thymine, and their nucleos(t)ides. This method is quite selective and approximately 20-fold more sensitive than the fluorimetric one; the detection limits for guanine nucleos(t)ides are 4–19 pmol ml −1 in the reaction mixture.

High-performance liquid chromatography of N-terminal tryptophan-containing peptides with precolumn fluorescence derivatization with glyoxal

A precolumn fluorescence derivatization method combined with high-performance liquid chromatography is described for the sensitive and selective determination of N-terminal tryptophan-containing peptides. The peptides and tryptophan were converted into fluorescent derivatives with glyoxal in a moderately acidic medium (pH 4.5). The derivatives were separated on a reversed-phase column with isocratic elution with an aqueous mobile phase composed of acetonitrile, methanol and phosphate buffer (pH 6.0), and subsequently detected by fluorimetry. The derivatization technique provided the respective N-terminal tryptophan-containing oligopeptides with single fluorescent peaks in chromatography. The detection limits for the peptides were 55-382 fmol per 100-microliters injection volume at a signal-to-noise ratio of 3. The method also allowed the facile detection of an N-terminal tryptophyl fragment in the enzyme reaction mixture of dynorphin A with trypsin.

Selective determination of guanine and its nucleosides and nucleotides by reaction with phenylglyoxal as a fluorogenic reagent

A fluorimetric method is described for determining guanine in its nucleosides and nucleotides. The method is based on the reaction of the compounds with phenylglyoxal as a fluorogenic reagent in a weakly acidic solution (pH 4.0). The fluorescences produced show excitation and emission maxima around 365 and 510 nm, respectively. The conditions established for the reaction do not produce fluorescence from other nucleic acid bases such as adenine, cytosine, uracil and thymine, and their nucleosides and nucleotides. The method is sensitive and selective for guanine and its derivatives, with a detection limit of 47–310 pmol ml−1 in the reaction mixture.

Determination of guanine and its nucleosides and nucleotides in human erythrocytes by high-performance liquid chromatography with postcolumn fluorescence derivatization using phenylglyoxal reagent

A high-performance liquid chromatographic method with on-line postcolumn fluorescence derivatization is described for the simple and sensitive determination of guanine and its nucleosides and nucleotides in human erythrocytes. After deproteinization of the biospecimen, guanine and its nucleosides and nucleotides were separated on a reversed-phase column (TSKgel ODS-120T) by gradient elution with methanol in the aqueous mobile phase consisting of tetra-n-propylammonium phosphate (pH 6.0) and phosphate buffer (pH 6.0). The compounds were then automatically converted into fluorescent derivatives by reaction with phenylglyoxal. This derivatization was selective for guanine-containing compounds. The present method permitted the reliable quantification of GDP and GTP in human erythrocytes. The detection limits (at a signal-to-noise ratio of 3) for guanine and its nucleosides and nucleotides were 3.2-10.0 pmol in a 20-microliters injection volume. The concentrations of GDP and GTP in human erythrocytes were 17.2 +/- 6.2 and 40.2 +/- 5.8 nmol/ml, respectively.

High-performance liquid chromatography of guanine and its nucleosides and nucleotides by pre-column fluorescence derivatization with phenylglyoxal reagent

Abstract A pre-column fluorescence derivatization method for the high-performance liquid chromatographic determination of guanine and its nucleosides and nucleotides is described. The compounds are converted into fluorescent derivatives by reaction with phenylglyoxal in a phosphate buffer (pH 6.0) at 37°C for 15 min. The derivatives are separated on a reversed-phase column (TSKgel ODS-120T) by gradient elution of acetonitrile in mobile phase containing 5 m M phosphate buffer (pH 6.5), and then detected fluorimetrically. The derivatization results in the guanine-containing compounds eluting as single fluorescent peaks. The method is highly selective and sensitive; the limits of detection for the compounds tested are 140–720 fmol per 100-μl injection volume.

Spectrofluorimetric assay for acyl CoA-cholesterol acyltransferase

Abstract A sensitive assay for acyl CoA-cholesterol acyltransferase (EC 2.3.1.26) in rat liver microsomal and mitochondrial preparations is described. The lowered cholesterol concentration in the enzyme reaction with oleoyl CoA is determined spectrofluorimetrically by using the cholesterol oxidase/peroxidase/ p -hydroxyphenylpropionic acid system. The assay requires as little as 20 μg of protein in the enzyme preparation.

A fluorimetric determination of lecithin-cholesterol acyltransferase in blood plasma

Abstract A fluorimetric assay for lecithin-cholesterol acyltransferase in human plasma (normal and pathological) is described. The lowered cholesterol concentration in the enzyme reaction is determined fluorimetrically by using the cholesterol oxidase—peroxidase —tyramine system. The assay requires only 10 μl of plasma.

Studies on Tetralin Derivatives. III

Ten kinds of hydroxytetralone oximes have been synthesized and their reactions with metallic ions have been investigated. It was found that the tetralone oxime having a hydroxyl group at the 8-position reacted with Cu2+, Ni2+, and Co2+, etc., forming insoluble, colored precipitates; but those containing no hydroxyl group at the 8-position or those having inactivated 8-methoxy or 8-acetoxy groups did not form the precipitate. The precipitate was found to be an intramolecular complex salt of the metal and the oxime by analysis of the compound and from the solubility of the precipitate in chloroform. 8-Hydroxy-5-methoxytetralone oxime had an especially sensitive reaction with metallic ions, and this could be utilized as a spot test reagent. Also, the chloroform solution of the complex salt of this oxime with Cu2+ and Ni2+ gave light yellow and light green colorations withmaximum absorption at 378 mμ and 395 mμ, respectively. The light absorbancy and the ion concentration were linearly related, and this relationship could be utilized in colorimetric determin ations of Cu2+. and Ni2+. The complex salt of Co2+ had an orange color and showed maximum absor ption at 372.5 mμ; but it changed gradually to dark brown, and the absorption finally became obscure.