Abstract
Abstract A selective adsorbent for the purification of α-galactosidases was prepared by attaching the ligand p-aminophenyl-melibioside by an amide linkage to succinoylaminoalkyl agarose. Small columns packed with this adsorbent quantitatively extracted ceramide trihexosidase activity and other α-galactosidases from plasma and partially purified fractions of these enzymes. Quantitative recovery of the enzymatic activity was readily achieved by the addition of detergent to the eluting buffer. The affinity chromatographic behavior of multiple forms of human ceramide trihexosidase activity has been investigated in plasma, kidney, and urine without prior purification of the enzymes. A total of five different forms of ceramide trihexosidase was observed in normal plasma, along with six forms of α-galactosidase that were specific for p-nitrophenyl-α-galactoside. The same α-galactosidase pattern was observed with urine concentrates, whereas cholate-solubilized lysosomal enzymes from kidney gave a simpler pattern with only one form of ceramide trihexosidase. The three plasma ceramide trihexosidases having optimal activity at pH 7.2 (B forms) were absent in plasma from heterozygous and hemizygous patients with Fabry's disease. Two forms of plasma ceramide trihexosidase with a pH optimum at 5.4 (A forms) were diminished in heterozygotes and were less than 5% of normal activity in plasma from hemizygotes. Only one of the six nonspecific α-galactosidases was deficient in Fabry plasma after affinity chromatography. Several additional proteins with affinity for the ligand were obtained from the plasma of Fabry patients. It is postulated that these proteins are catalytically inactive forms of ceramide trihexosidase.
Highlights
A selective adsorbent for the purification of a-galactosidases was prepared by attaching the ligand p-aminophenylmelibioside by an amide linkage to succinoylaminoalkyl agarose
Adsorbent-p-Aminophenylmelibioside was synthesized in five steps from melibiose in an over-all yield of about 45 ‘%
Glucose, and pnitrophenol or p-aminophenol were liberated from the p-nitrophenyl- and p-aminophenylmelibiosides by acid hydrolysis, confirming that partial hydrolysis of the disaccharide moiety had not occurred during the synthesis
Summary
Preparation of Afinity Column AdsorbentXelibiose Octaacetate-Acetylation of melibiose and conversion of the product to the bromo derivative were carried out by modification of the procedures described by Wolfrom and Thompson [19]. Acetic anhydride (50 ml) and dry pyridine (50 ml) were added to 5 g of melibiose. The suspension was heated at 80” until the carbohydrate dissolved, and the solution was allowed to stand at room temperature for 12 hours. Dry methanol (100 ml) was added slowly with stirring and allowed to stand for 3 hours. The solution was evaporated to dryness uuder reduced pressure at 603, and the product was dried in vacua over potassium hydroxide pellets. Dcstobromomelibiose-Melibiose octaacetate (8.8 g) was dissolved in 50 ml of a freshly prepared solution of acetic anhydride containiug 40% hydrogen bromide. Final traces of acetic acid were removed by drying the residue in vacua over potassium hydroxide pellets
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