Fluorescence lifetimes of haem proteins excited into the tryptophan absorption band with synchrotron radiation.

Abstract

FLUORESCENT spectra of tryptophan residues in proteins are a powerful tool in the study of the intraprotein medium, primarily as an indicator of local conformational changes of the residue microenvironment. Fluorescence quantum yields of tryptophan in the case of haem proteins, however, are very small (Q ≤0.002)1, and a dramatic increase (Q′ ∼ 0.2) is observed after haem removal. The photodissociation of carbon monoxymyoglobin, when irradiated using ultraviolet light, has indicated the transfer of excitation from the aromatic amino acid to the haem group2,3 following a mechanism of the Forster type4; this transfer has a probability1 at least 100 times greater than the fluorescence, and therefore some 20 times greater than the other competing radiation-free processes. Consequently lifetimes have been considered to be extremely small and no attempts have therefore been made to measure them. This type of information is, however, vital for our understanding of the quenching mechanism by haem. We have therefore reinvestigated the problem with respect to decay times for ox, horse and adult human haemoglobins and their α and β subunits when excited in the tryptophan absorption band. Our data are not refined—lifetime values reported here have a rather poor precision (∼ ±0.25 ns)—but those obtained using ox, horse and human samples demonstrate similar behaviour. In some selected cases data from haemoglobin and apohaemoglobin have been compared.