orabase and 4-hydroxymercuribenzoate

Conformation and microenvironment at the active site of 1,4-beta-D-glucan glucanohydrolase was probed with fluorescent chemo-affinity labeling using o-phthalaldehyde. OPTA has been known to form a fluorescent isoindole derivative by cross-linking the proximal thiol and amino groups of cysteine and lysine. Modification of lysine of the enzyme by TNBS and of cysteine residue by PHMB abolished the ability of the enzyme to form an isoindole derivative with OPTA. Kinetic analysis of the TNBS and PHMB-modified enzyme suggested the presence of essential lysine and cysteine residues, respectively, at the active site of the enzyme. The substrate protection of the enzyme with carboxymethylcellulose (CMC) confirmed the involvement of lysine and cysteine residues in the active site of the enzyme. Multiple sequence alignment of peptides obtained by tryptic digestion of the enzyme showed cysteine is one of the conserved amino acids corroborating the chemical modification studies.

Topics: Actinomycetales; Binding Sites; Carboxymethylcellulose Sodium; Circular Dichroism; Cysteine; Dose-Response Relationship, Drug; Enzyme Activation; Glucan 1,4-beta-Glucosidase; Hydroxymercuribenzoates; Kinetics; Lysine; Molecular Sequence Data; Molecular Weight; Protein Conformation; Sequence Alignment; Substrate Specificity; Trinitrobenzenesulfonic Acid

A rapid method for hemoglobin chain recombination which gives a homogeneous product was developed. The method utilizes a small carboxymethylcellulose column as a medium for chain recombination and concentration of the hemoglobin. Equimolar amounts of p-hydroxymercuribenzoate derivatives of alpha- and beta-chains were mixed with 300 x molar excess of beta-mercaptoethanol over the p-hydroxymercuribenzoate groups. After 10 min of incubation in an ice bath, the mixture was adjusted to pH 5.85, and was loaded on a carboxymethylcellulose column. The column was washed with 10 mM phosphate buffer-1 mM Na2EDTA-47 mM beta-mercaptoethanol, pH 5.85 and then with 10 mM phosphate buffer, pH 5.85. The hemoglobin was eluted from the column by use of 15 mM K2HPO4. The hemoglobin was homogeneous on polyacrylamide gel electrophoresis and had a visible spectrum, electrophoretic mobility, and number of -SH groups comparable to those shown by control hemoglobin.

Topics: Carboxymethylcellulose Sodium; Chromatography; Electrophoresis, Polyacrylamide Gel; Hemoglobins; Humans; Hydroxymercuribenzoates; Indicators and Reagents; Mercaptoethanol; Protein Conformation