Hydrated electron formation on laser excitation of P-pyridoxyl amino acids and proteins.

Evidence from a characteristic transient spectrum, its lifetime and quenching by N2O demonstrates that XeCl excimer laser excitation of the reduced form of pyridoxal 5'-phosphate bound to lysine residues will produce the hydrated electron. The phenoxyl-type radical coproduct was also observed. The results identify a useful label of protein structures for site-specific electron generation which can occur outside the region of light absorption by aromatic residues of protein. The results also represent the first observation of hydrated electron formation from flash excitation of a vitamin B6 cofactor.

The Fourier transforms of the laser-induced absorption decay from glycogen phosphorylase and DOPA decarboxylase.

Fourier analysis of the laser-induced absorption decay curves of 3,4-dihydroxyphenylalanine (DOPA) decarboxylase and glycogen phosphorylase demonstrates a powerful technique in the analysis of complicated decay behavior. Phosphorylase which uses the pyridoxal 5'-phosphate cofactor in an unknown manner exhibits over weak absorption an intense decay while decarboxylase demonstrates only weak absorption. Fourier analysis of the decay curves clearly shows that phosphorylase has an intense absorption decay in the midst of three weaker ones and that decarboxylase only has three weak decays. This conclusion justifies the isolation and use of the intense decay of phosphorylase as an observable in the study of protein dynamics at the active site about the cofactor. The decay has demonstrated a movement of positive charge to substrate in the mechanism of phosphorylation of glycogen units.

Fluorescence lifetime and polarization anisotropy studies of membrane surfaces with pyridoxal 5'-phosphate.

The fluorescence lifetime and depolarization of the pyridoxal 5'-phosphate label demonstrated different environments at the structure-solvent interface for micelles, liposomes, proteins and membranes. A short lifetime and rotational correlation time for the micelles and liposomes proved that the label was strongly associated with the water solvent and rotated freely about the covalent bond. The proteins provided a more buried or hydrophobic site as shown by an increase in the lifetimes. Rotational correlation times of 4-6 ns for sarcolemma and erythrocyte membranes suggested restricted rotation for the pyridoxal 5'-phosphate label. Lower values of the rotational correlation time for rod outer segment and myelin sheath proved that the protein epsilon-amino groups are at the solvent interface which allows for more rotation.

Mathematical separation of multi-component exponential signals from the u.v. laser excitation of glycogen phosphorylase b.

Laser excitation of the vitamin B6 cofactor of the glycogen phosphorylase enzyme produces a transient absorbance signal at 470 nm. Martin et al. proposed four exponential decays for this complex signal. One component with the largest amplitude and a decay rate constant in the region of 150,000 s-1 results from an excited singlet state, and three successive decays of smaller amplitude with the rate constants in the regions of 700,000 s-1, 30,000 s-1, and 6000 s-1 result from a triplet state. These results were determined through nonlinear least squares regression and residual analyses, with some knowledge of the possible photochemistry of the cofactor by itself. The Fourier transform method, which requires no initial estimates of the parameters or of the number of decays, was selected for further analysis of the data. The results of the Gardner and differential approaches to the method confirm that the predicted four exponential components are in the signal and that the values of the decay rate constants agree with those from the nonlinear regression analysis. These results, presented here, help to demonstrate protein changes at the active site of enzyme catalysis.

Protein dynamics of glycogen phosphorylase.

The glycogen phosphorylase molecule absorbs the ultraviolet energy of a nitrogen laser to form an excited state of the cofactor. The decay rate of this state has a lifetime of 6.7 microseconds, and its sensitivity to bound substrates presents a new perspective of the mechanism. A careful analysis of the decay curve for native enzyme and cofactor analogues showed that the lifetime depends on the conformation of protein groups at the active site and how the residues change with bound substrate. The reactive ternary complexes obtained from either direction of the reaction yielded the same lifetime, indicating a change in the active-site conformation to a common configuration for the cofactor and substrate phosphate. This configuration indicates an increase in the cofactor 5'-PO4 pKa and a possible proton shuttle. The pyridoxal 5'-pyrophosphate reconstituted enzyme showed no conformational change alone or in the presence of oligosaccharide. This result does not support an electrophilic attack by the 5'-PO4 phosphorus.

Interactions at the active site of glycogen phosphorylase b. A new laser probe.

The flash excitation of the pyridoxal 5'-phosphate cofactor of glycogen phosphorylase b by an ultraviolet laser produces a transient state from a proton transfer of the bound cofactor. The rate of decay of this transient state is sensitive to the ionization state of the cofactor. This proved a useful probe for the ionization state of the 5'-phosphate group of the cofactor on the binding by the enzyme of various substrates. The decay rate data show, for the binding of glucose 1-phosphate, a partially negative 5'-HPO4- and evidence for a PO4-PO4 interaction. The data is interpreted in terms of a dynamic shift of substrates at the active site.