COLORIMETRIC ASSAY FOR LECITHIN USING 2 CO-IMMOBILIZED ENZYMES AND ANINDICATOR DYE CONJUGATE

According to the new assay for lecithin, phospholipase D (PL), choline oxidase (ChOX), and the indicator conjugate bromothymol blue-glutathi one (BTB-GSH) are co-immobilized on glutaraldehyde-activated polyacryl amide transparent gel. When a sample containing lecithin is added to t he biocatalytic gel, lecithin is hydrolyzed by PL to choline, which is oxidized to betaine by ChOX. The organic acid betaine changes the mic roenvironmental pH of the enzymes, thus leading the optical absorption of the biocatalytic gel to a hypsochromic shift because of the co-imm obilized indicator conjugate, BTB-GSH. The change of the optical absor ption at lambda(max) (622 nm) is related linearly to lecithin concentr ation in the range 20-120 mu M. When the optical effect is quantified in a spectrophotometer, the analyte concentration can be determined. T he biocatalytic gel is used successfully for lecithin determinations i n bovine serum and soybean granules. The principle of the method is ap plicable to the construction of optical biosensors.