The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis

The Carbon Dioxide Hydration Activity of Carbonic Anhydrase I. STOP-FLOW KINETIC STUDIES ON THE NATIVE HUMAN ISOENZYMES B AND C

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Multiple Binding Modes of Inhibitors to Carbonic Anhydrases: How to Design Specific Drugs Targeting 15 Different Isoforms?

Esterase Activities of Human Carbonic Anhydrases B and C

Quenching of the tryptophan fluorescence of na- tive proteins was studied using oxygen concentrations up to 0.13 M, corresponding to equilibration with oxygen at a pres- sure of 1500 psi. Measurement of absorption spectra and en- zymic activities of protein solutions under these conditions reveal no significant perturbation of the protein structure. The oxygen quenching constant (k+*) for a variety of proteins indi- cates that the apparent oxygen diffusion rate through the pro- tein matrix is 20-50z of its diffusion rate in water. No tryp- tophan residues appear to be excluded from quenching, and no correlation of the fluorescence emission maxima with k+* T he previous paper (Lakowicz and Weber, 1973) de- scribed the methodology and presented experimental data for the quenching of small molecules, and some linear biopoly- mers, by oxygen. Here we examine the quenching of the fluo- rescence of proteins by oxygen. X-Ray determined structures and solvent perturbation studies of many proteins have shown that tryptophan residues are often situated in the interior of the protein matrix and appear inaccessible to solvent. It must be pointed out, however, that both of these techniques yield information about the average conformation and solvation of the amino acid residues, but no information about the ex- istence of the structural fluctuations which may occur. Since quenching of fluorescence by oxygen depends on the colli- sional rate between oxygen and fluorophore, we expected oxygen quenching of tryptophan fluorescence in proteins to yield information on the dynamics of those structural changes in the nanosecond time scale that would allow diffusion of oxygen through the protein matrix. Such local fluctuations must be indispensable for the effective quenching of a fluoro- phore shown by X-ray structural studies to be out of direct contact with water. The quenching of the fluorescence of fluorophore residues forming part of a protein, or in general of a compact macro- molecule, presents many complexities that are absent in the case of an isolated fluorophore in solution. These complexities can best be examined by reference to the modified Stern- Volmer equation discussed in the previous paper

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Jacob A. Verpoorte

Papers

Esterase Activities of Human Carbonic Anhydrases B and C

Purification and Properties of Human Erythrocyte Carbonic Anhydrases

Nitration and iodination of human carbonic anhydrases. Effects on enzyme activity and on optical properties.