Determination of meso-Alanopine and D-Strombine by High Pressure Liquid Chromatography in Extracts from Marine Invertebrates

Abstract

Meso-Alanopine and D-strombine are separated by high pressure liquid chromatography using a cation exchange resin and 2.5 .times. 10-5 M sulfuric acid as eluant, at a flow rate of 1.0 ml/min, 20.degree. C column temperature and a pressure of 4500 kPa [Pascal]. Both opines were detected by conductivity. Separation and quantitation was possible in the range of 0.05-25 nmol of meso-alanopine and D-strombine. Chemically or enzymatically synthesized opines were quantitated using alanopine/strombine dehydrogenase from Crassostrea angulata. The enzyme was purified by ammonium sulfate precipitation, Sephadex G-100 filtration and fast-protein-liquid chromatography. Specific activity of the final preparation was 500 U/mg protein with glycine as substrate. The formation of meso-alanopine and D-strombine was demonstrated in neutralized perchloric acid extracts from muscle tissue of Arenicola marina L. following enhanced muscular activity and in Mytilus edulis L., Nucula nitida and C. angulata after 24 h of anoxia.