Purification of mammalian arylsulfatase A enzymes by subunit affinity chromatography.
Rabbit liver arylsulfatase A (arylsulfatase sulfohydrolase, EC 3.1.6.1) monomer was immobilized on cyanogen bromide-activated Sepharose-6MB and on Affi-Gel-10 under various experimental conditions in order to study the effects of variables in sulfatase monomer/oligomer subunit affinity chromatography. First, the number of reactive groups on activated Sepharose-6MB and Affi-Gel-10 was determined by a procedure involving spectrophotometric titration with L-tyrosine. After covalent coupling of sulfatase monomers to the gels, the enzyme binding capacities of the sulfatase subunit affinity gel matrixes were determined at pH 4.5. The maximum binding of free monomers from solution could be achieved when the Affi-Gel-10 protein monomer matrix was prepared at low degrees of covalent loading. The introduction of a batch technique for equilibration of the protein sample with the monomer affinity matrix also increased the efficiency of the subunit affinity gel in purification procedures. The effect of pH on the stability of the heterodimers formed between monomers of rabbit liver arylsulfatase A immobilized on Affi-Gel-10 and free monomers of arylsulfatase A enzymes from different tissues and organisms was studied using the batch technique. For all sulfatase A enzymes tested, the midpoint of the pH transition for subunit association was pH 6.2, suggesting that the amino acid residues involved in the dimerization are similar. The versatility of the Affi-Gel-10 monomer affinity matrix was further demonstrated by purifying 13 mammalian arylsulfatase A enzymes to homogeneity, as assessed by Sephacryl chromatography, native and SDS gel electrophoresis. The molecular weights of the homogeneous monomers and their peptide subunits were in the range of 110-180 KDa and 50-64 KDa, respectively. The amino acid compositions of these enzymes were also determined.
Waheed
A
A
Van Etten
R L
RL
D000818:Animals; D002417:Cattle; D002553:Cerebroside-Sulfatase / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D005260:Female; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008297:Male; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D013045:Species Specificity; D013429:Sulfatases / Q000302:isolation & purification*; D013737:Testis / Q000201:enzymology; D014018:Tissue Distribution
D046911:Macromolecular Substances; D013429:Sulfatases; D002553:Cerebroside-Sulfatase