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Biomedical subjects

B M Dunn

Publications and source records attributed to B M Dunn.

119 records · Page 7Linked to original sources
PubMed1975

Evaluation of quantitative affinity chromatography by comparison with kinetic and equilibrium dialysis methods for the analysis of nucleotide binding to staphylococcal nuclease.

The elution of staphylococcal nuclease on thymidine 3'-(p-Sepharose-aminophenyl phosphate) 5'-phosphate (nucleotide ligand of nuclease covalently bound to Sepharose 4B) was studied in the presence of a variety of soluble nucleotide ligands. The elution volumes of nuclease vary proportionally with matrix-bound ligand concentration (at constant soluble ligand concentration), inversely with soluble ligand concentration (at constant matrix-bound ligand concentration), and inversely with dissociation constant of soluble ligand (at constant concentrations of soluble and matrix-bound ligand). The variation of elution volume was related to an expression which described the competition of soluble and matrix-bound ligand for nuclease binding. Using this expression, values for dissociation constants were derived for nucleotide ligands in both the soluble and bound form. The values for soluble ligand were found to correspond closely to those obtained by either equilibrium dialysis or kinetics of inhibition of nuclease activity. Furthermore, a close correspondence was found between the values of dissocation constants for matrix-bound and soluble thymidine 3'-(p-aminophenyl phosphate) 5'-phosphate, thus defining the interaction of nuclease with the matrix-bound ligand as a process quite similar to that occurring in solution.

Chromatography, Affinity↗
PubMed1974

Quantitative affinity chromatography. Determination of binding constants by elution with competitive inhibitors.

The affinity chromatographic separation procedure was tested for its utility in quantitating the affinity of proteins to both insolubilized ligand and corresponding competing soluble ligand. An expression has been derived that (1) allows the determination of binding constants for the interaction of a soluble protein species with an affinity chromatography matrix involving active site binding, (2) yields binding constants for the interaction of the protein with soluble ligands that can compete with the insoluble matrix for binding to the active site of the protein, and (3) utilizes readily obtainable parameters from conventional chromatography. This expression has been applied to the quantitation of the binding of staphylococcal nuclease to thymidine-5'-phosphate-3'-amino-phenylphosphate-Sepharose in competition with thymidine-3',5'-bisphosphate.

Bacterial Proteins↗
PubMedDate not supplied

Synthesis and biological activity of six monosubstituted analogs of a sea anemone polypeptide neurotoxin.

Monosubstituted analogs of Stichodactyla helianthus neurotoxin I (ShI), a sea anemone polypeptide, were synthesized by solid-phase methods. The analogs were selected to probe the importance of charged residues in the amino-terminal region; ionizable side chains were replaced with a corresponding neutral isosteric amino acid side chain. Following oxidation of the three disulfide bonds and refolding, the analogs were purified by gel, ion-exchange and reversed-phase chromatography. Circular dichroism and fluorescence spectral analyses indicate that the analogs have folded structures very similar to the native toxin. Crustacean paralytic bioassays and neuronal receptor binding assays of ShI analogs show that the anionic triad Asp 6-Asp 7-Glu 8 is essential for activity, as replacement of any of these side chains with the corresponding amide reduces toxicity and binding at least 1000-fold. Substitution at Lys 4 or Asp 11 reduces biological activity at least 10-fold; these residues are apparently not as critical as the polyanionic region. Replacing the N-terminal Ala residue with a Tyr residue has only a slight effect upon crab toxicity and axolemma binding. Our results suggest that several amino acid side chains near the N-terminus of this sea anemone neurotoxin are important for binding to crab neuronal sodium channels.

Amino Acid Sequence↗