Depolarization of the intrinsic and extrinsic fluorescence of pepsinogen and pepsin.
1. The effects on the intrinsic tryptophan emission anisotropy of pepsin and pepsinogen solutions produced by (a) changes in temperature, (b) increases in viscosity with added glycerol at constant temperature and (c) decreases in lifetime through collisional quenching by potassium iodide were measured at several excitation wavelengths. The rotational-relaxation times calculated from results provided by method (b) approximate to the theoretical values for the two proteins, on taking hydration and shape factors into account, on the basis of random orientation of the tryptophan groups within the macromolecules. Differences between the results provided by methods (b) and (c) are attributable to inter-tryptophan resonance-energy-transfer depolarization, and the anomalous values recorded in method (a) can be attributed to the temperature-dependence of the limiting anisotropies. 2. Two different monomeric conjugates of pepsin, each containing one extrinsic fluorescent group per macromolecule, gave widely different relaxation times. This difference may arise from a specific orientation of the emission dipole in the enzyme. In active-site-labelled pepsin (1-dimethylaminonaphthalene-5-sulphonylphenylalanine-pepsin) this orientation would be approximately parallel to the symmetry axis of the equivalent ellipsoid, whereas in the other conjugate (1-dimethylaminonaphthalene-5-sulphonyl-pepsin) the orientation may be roughly normal to this direction, or some independent rotation of parts of the protein molecule is possible.
D001665:Binding Sites; D004563:Electrochemistry; D004792:Enzyme Precursors; D005453:Fluorescence; D005990:Glycerol; D009281:Naphthalenes; D010434:Pepsin A; D010649:Phenylalanine; D011193:Potassium Iodide; D013053:Spectrophotometry; D013451:Sulfonic Acids; D013696:Temperature; D014364:Tryptophan; D014783:Viscosity
D004792:Enzyme Precursors; D009281:Naphthalenes; D013451:Sulfonic Acids; D011193:Potassium Iodide; D010649:Phenylalanine; D014364:Tryptophan; D010434:Pepsin A; D005990:Glycerol