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