Ealthcare). Analytical procedures and enzyme characterization. (i) Electrophoretic analysis. SDS-PAGE was
Ealthcare). Analytical approaches and enzyme characterization. (i) Electrophoretic evaluation. SDS-PAGE was performed on five to 15 polyacrylamide gradient gels with Coomassie brilliant blue R250 or silver staining as described by Laemmli (30). The relative molecular mass of the purified catalase was estimated based on the molecular mass of protein markers (GE Healthcare). (ii) Isoelectrophoresis. The isoelectric point of catalase A1 was determined by isoelectric focusing (IEF) on precast gels LKB-IEF (three.5 to 9.5 and 4 to six.5; GE Healthcare). Just after completion of electrophoresis, the gels had been incubated for 20 min within a 1 mM option of horseradish peroxidase in PBS, and hydrogen peroxide was added at a final concentration of five mM. After Amphiregulin Protein Molecular Weight incubation for 10 min, washing in distilled water, and addition of 2 mM three,3=-diaminobenzidine (DAB) in PBS, catalases appeared as unstained regions on a brown background. The pI was extrapolated from the migration of isoelectric point markers from GE Healthcare. (iii) Impact of pH and temperature on catalase activity. The pH stability of the catalase was determined by measuring the catalase activity within a range of pH (two.five to 13) using 0.2 M sodium acetate buffer (pH 2.five to four.five), 66 mM sodium potassium phosphate buffer (pH five to eight), or 0.1 M glycine buffer (pH 9 to 13). Heat stability was evaluated by measuring the residual enzyme activity right after 1 to 15 min of incubation at various temperatures (37, 68, 80, and 100 ). The residual catalase activity was determined by densitometric determination just after native Page and negative staining on the gels. (iv) Catalytic properties on the catalase. The effects of many catalase inhibitors had been evaluated by UV spectrophotometry just after incubation for 1 h with all the purified enzyme (Table 1). Inhibitors of hemoproteins such as potassium cyanide (KCN) and sodium azide (NaN3) have been tested at ten mM final concentrations, whereas 3-amino-1,two,4-triazole (3-AT), a precise inhibitor of catalase, was tested at a four mM final concentration. In addition, the effects of metallic ions Cu2 and Hg2 (ten mM), SDS (four ), and 2-mercaptoethanol (2-ME) (30 mM) have been also evaluated. Stability on the enzyme in ethanol-chloroform was tested as described by Nadler et al. (31). (v) Glycosylation. Glycosylation of catalase A1 was very first investigated by affinity chromatography on a FGF-2 Protein custom synthesis concanavalin A (ConA)-conjugated Sepharose 4B column (GE Healthcare). Two hundred microliters of the crude extract was incubated for 30 min at 37 with ConA-Sepharose. Immediately after centrifugation for 5 min at 4,000 g and washing in PBS, glycosylated proteins had been eluted with 0.2 M methyl -D-mannopyranoside in PBS. Soon after a further 30-min incubation at 37 and centrifugation, the unbound fraction and eluted proteins had been analyzed for catalase activity by native Web page and damaging staining. Glycosylation was also investigated soon after electrophoretic transfer of proteins separated by SDS-PAGE on a Hybond-P membrane (GE Healthcare). Soon after electrotransfer, the membrane was blocked overnight at four with five bovine serum albumin (BSA) in PBS, washed three occasions with PBS, and then incubated for 1 h at 37 with peroxidase-conjugated wheat germ agglutinin (WGA) (1 gml) or ConA (3 gml) from SigmaAldrich in 50 mM Tris buffer supplemented with 0.1 mM Ca2 and 0.1 mM Mg2 . Following washing, peroxidase was revealed with 0.5 mgml DAB in 0.1 M Tris buffer (pH 7.6) and 0.1 hydrogen peroxide. Human sera. A panel of 64 human serum samples was applied to evaluate the usefu.
