Thin layer chromatography/fluorescence detection of 3,4-methylenedioxy-methamphetamine and related compounds.

A rapid and sensitive method for the detection of six methylenedioxylatedphenethylamines, 3,4-methylenedioxymethamphetamine (MDMA); 3,4-methylenedioxyamphetamine; 3,4-methylenedioxyethylamphetamine; N-methyl-1-(3,4-methylenedioxyphenyl)-2-butamine; N-methyl-1-(3,4-methylenedioxyphenyl)-3-butamine; and 3,4-methylenedioxydimethylamphetamine, by thin-layer chromatography with fluorescence detection is proposed. These compounds form fluorophores on the developing plate following spraying with a reagent consisting of sodium hypochlorite, potassium hexacyanoferrate (III), and sodium hydroxide, and heating for 3 min at 100 degrees C. Blue fluorescent spots were observed under ultraviolet light in a wavelength range of 250-400 nm. The detection limits for MDMA and the above related compounds were 50 ng. The proposed method was effectively applied to the detection of MDMA in urine samples.

Micelle formation of bile salts and zwitterionic derivative as studied by two-dimensional NMR spectroscopy.

The self-association of sodium taurodeoxycholate (NaTDC) and a zwitterionic derivative of cholic acid (CHAPS) in deuterium oxide was investigated by one- and two-dimensional nuclear magnetic resonance spectroscopy (NMR) spectroscopy. Analysis of the concentration dependence of the chemical shifts of several protons suggested that NaTDC and CHAPS form nonamers and heptamers, respectively, as well as dimer. The equilibrium constants of dimerization and the micellar aggregation numbers are close to the literature values. From the intensities of intermolecular cross-peaks in the nuclear Overhauser effect spectroscopy (NOESY) and rotating frame nuclear Overhauser effect spectroscopy (ROESY) spectra of NaTDC and CHAPS micellar solutions, partial structures of their micelles were estimated. The CHAPS micelle consists mainly of the back-to-back association, similarly to taurocholate (NaTC). However, the NaTDC micelle consists of the back-to-face association, because the face of NaTDC is rather hydrophobic. Furthermore, the back of bile molecules forms a convex plane and the face forms a concave plane. The back-to-face structure of NaTDC will be stabilized by a close contact between these planes. The chemical shift changes of several protons of CHAPS and NaTC in the micellar state are close to each other, but are different from those of NaTDC. This finding is consistent with the difference in their micellar structures.

Heme reduction by intramolecular electron transfer in cysteine mutant myoglobin under carbon monoxide atmosphere.

Human myoglobin (Mb) possesses a unique cysteine (Cys110), whereas other mammalian Mbs do not. To investigate the effect of a cysteine residue on Mb, we introduced cysteine to various sites on the surface of sperm whale Mb (K56C, V66C, K96C, K102C, A125C, and A144C) by mutation. The cysteines were inserted near the end of alpha-helices, except for V66C, where the cysteine was introduced in the middle of an alpha-helix. Reduction of the heme was observed for each mutant metMb by incubation at 37 degrees C under carbon monoxide atmosphere, which was much faster than reduction of wild-type metMb under the same condition. Heme reduction did not occur significantly under nitrogen or oxygen atmospheres. The rate constant for heme reduction increased for higher mutant Mb concentration, whereas it did not change significantly when the CO concentration was reduced from 100% CO to 50% CO with 50% O(2). The similarity in the rate constants with different CO concentrations indicates that CO stabilizes the reduced heme by coordination to the heme iron. SDS-PAGE analysis showed that mutant Mb dimers were formed by incubation under CO atmosphere but not under air. These dimers were converted back to Mb monomers by an addition of 2-mercaptoethanol, which showed formation of a Mb dimer through a disulfide bond. The rate constant decreased in general as the heme-cysteine distance was increased, although V66C Mb exhibited a very small rate constant. Since V66 is placed in the middle of an alpha-helix, steric hindrance would occur and prevent formation of a dimer when the cysteine residues of two different V66C Mb molecules interact with each other. The rate constants also decreased for K56C and A144C Mbs presumably because of the electrostatic repulsion during dimer formation, since they are relatively charged around the inserted cysteine.