Neutralized Phthalate Buffer

The incubation mixture contained in a Anal volume of 100 /iL 400 fiM DL-homocysteine, 500 (iM ( )-L-A/5-methyltetrahydrofolate, 50 fiM cyanocobalamin, 300 fiM 5-adenosylmethionine, 125 mM 2-mercaptoethanol, 20 fiM L-norvaline, 50 mM potassium phosphate buffer (pH 7.4), and 50 /xL of liver or cell extract. The incubation mixture was immediately flushed with nitrogen and overlayered with 50 /tL of bis(3,5,5-trimethylcyclohexyl)-phthalate. The incubation, carried out at 37°C in the dark, was stopped by the addition of 10 / L of 4 TV perchloric acid. The acid was then neutralized by addition of 10 / L of 4 TV KOH containing 3.3 M potassium bicarbonate. After centrifugation, 90 /iL of supernate was mixed with 175 fiL of o-phthaldialdehyde reagent (prepared by mixing 1 mL of 56 mM o-phthaldialdehyde in methanol with 9 mL of 0.1 M sodium borate buffer, pH 9.5, then adding 40 fiL of 2-mercaptoethanol). After 2 minutes at 23°C, 220 /xL of this mixture was used for HPLC analysis. The assay is linear for at least 2 hours. 

The protein polysaccharide is hydrolyzed at 100-110° for 3 hours with N sulfuric acid, neutralized with baryta solution, filtered, and the filtrate concentrated to small volume. Zone electrophoresis in borate buffer (pH 8.6) is then carried out for a suitable time, and the paper strip is dried and sprayed with ninhydrin (to locate the amino acids) and with aniline hydrogen phthalate (to detect the reducing sugars). 

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