Complex photophysics of the single tryptophan of porcine pancreatic phospholipase A2, its zymogen, and an enzyme/micelle complex.
The fluorescence emission of the single tryptophan in porcine pancreatic phospholipase A2, its zymogen, and a micellar complex of the enzyme with the nonhydrolyzable substrate analogue n-hexadecylphosphocholine has been studied by both steady-state and time-resolved techniques. Stern-Volmer quenching studies with acrylamide indicate that, both in the enzyme and in the zymogen, the tryptophan is exposed to solvent. Similar studies with ionic quenchers show that there is appreciable ionic character to the tryptophan environment. Single photon counting fluorescence measurements were performed using a high repetition rate synchronously pumped dye laser as a light source. When tryptophan fluorescence is collected with a broad-band (80-nm) emission filter, the decay kinetics in the enzyme and the zymogen require at least three, and often four, exponential terms for a proper description. The decay kinetics can be adequately described by three exponential terms when the fluorescence is collected at specific wavelengths by using narrow (10-nm) band-pass filters. The lifetimes are approximately constant across the emission band, but the amplitudes vary with the fraction of the long lifetime increasing at longer emission wavelengths. Formation of a complex between phospholipase A2 and micelles of n-hexadecylphospohocholine produces large changes in the tryptophan emission that are associated with transfer to a hydrophobic environment. The decay kinetics of tryptophan in the enzyme/micelle complex appears to require only two exponential terms. This is the first reported instance of fluorescence data from a single tryptophan protein requiring more than double-exponential decay kinetics. The results are discussed in terms of the range of environments sampled by the tryptophan residue and the resulting distribution of lifetimes.
D000818:Animals; D007700:Kinetics; D008823:Micelles; D010179:Pancreas / Q000201:enzymology*; D010740:Phospholipases / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D010777:Photochemistry; D013050:Spectrometry, Fluorescence; D013552:Swine; D014364:Tryptophan / Q000032:analysis*
D008823:Micelles; D014364:Tryptophan; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2