Chromatographic purification of ribose 5-phosphate ketol isomerase from Candida utilis on p-mercuribenzoate-6-aminohexyl-sepharose 4B adsorbent.

Abstract

Candida utilis ribose 5-phosphate ketol isomerase has already been purified from cell-free extracts of C. utilis by the authors1) according to conventional pro cedures composed of ammonium sulfate precipitation, DEAE-cellulose (DE-23) column chromatography, Bio-Gel P-200 gel filtration and repeated preparative polyacrylamide gel electrophoresis.But, by this procedure, the enzyme yield was very low. So, in order to get a good yield of the enzyme preparation, purification procedure using affinity chro matography was tested. As an adsorbent for affinity chromatography, Sepharose 4B having p-mercuribenzo ate (p-MB) as a ligand and 6-amino-hexylamine as a spacer was used.Preparation of p-MB-6-aminohexyl-Sepharose 4B2) Sepharose 4B (25ml) suspended in distilled water (25ml) was activated by addition of cyanogen bromide (6.25g) at pH 11 -11.5. Cyanogen bromide-activated Sepharose 4B gel was swollen and washed successively with water (one liter), 0.1 M NaHCO3 (one liter) and finally with water (one liter). To the cyanogen bromi de-activated Sepharose 4B (25ml), 0.05 M 6-amino hexylamine (25ml) was added and adjusted pH at 10. After stirring at 4°C for 16 hr, it was filtered on a glass filter and successively washed with 0.1 M NaHCO3 and water. A half gram of p-MB to be coupled with 6-aminohexyl-Sepharose 4B was dissolved in 45ml of 40% dimethylformamide.To the mixture; 6-aminohexyl-Sepharose 4B (25ml) and 4 mm 1-ethyl-3-(3-dimethylaminopropyl) carbodi imide were added. After being adjusted pH to 6.0 with I N HCl, the mixture was kept at room temperature for 16 hr.After then, it was filtered with a glass filter and washed successively with 0.1 M NaHCO3 (one liter), 40 % dimethylformamide (500ml) and finally with water (one liter). Next, the column was washed successively with 0.1 % bovine serum albumin (250ml), 8 M urea in 0.1 M citrate buffer (pH 5.5, 250ml) to remove any trace of uncoupled p-MB, and then washed successively with 8 M urea in 0.1 M citrate buffer containing 50mm 2-mercaptoethanol (250ml), water (250ml), 40 % dime thylformamide (250 nil) and water (250 ml).Purification of C. utilis ribose 5-phosphate ketol isomerase by affinity chromatography on a p-MB-6-aminohexyl-Sepharose 4B was carried out in the fol lowing procedure. The adsorbent was equilibrated with 0.01 M Tris-HCl buffer (pH 7.3, hereafter referred as column buffer), and then 15.0 mg of partially purified ribose 5-phosphate ketol isomerase preparation (DE-23 eluate) dissolved in 70 ml of the column buffer, was applied to the column. After washing the column suc cessively with the column buffer and the column buffer containing 0.1 M NaCl, the column buffer containing 0.2 M NaCl was used to elute ribose 5-phosphate ketol isomerase from the column as shown in Fig. 1. Ribose 5-phosphate ketol isomerase (3.4mg) was recovered by this procedure. The purified enzyme preparation by affinity chromatographic method showed homogeneity on a polyacrylamide disc electrophoresis as shown in Fig. 2. The results of purification of ribose 5-pho sphate ketol isomera...