Bacterial luciferase catalyzes the oxidation of reduced flavin mononucleotide by molecular oxygen; long-chain aldehyde is required for light emission. At 200 the bioluminescence has a lifetime of tens of seconds, while excess reduced flavin is removed by way of nonenzymatic autoxidation in less than a second. This observation indicates the existence of a long-lived enzyme intermediate, which has been postulated to be a peroxide of the enzyme-bound reduced flavin. This intermediate was isolated and studied at low temperature (-20°) emission of light, an increase in both absorption (450 nm) and fluorescence emission (530 nm) occurred.The most important requirement for a detailed study of the enzyme intermediate is that it be separated from the free flavin. In recent years, techniques for studying enzymes and their reactions at low temperatures have been developed in our laboratory (3). Since the lifetime of intermediate II at low temperatures (-20°to -50°) is measured in hours or days (4), separation by column chromatography at -20°was attempted and achieved. Intermediate II was found to have an absorption maximum at 370 nm and a fluorescence emission peaking at 485 nm, consistent with the hypothesis that it involves a flavin-peroxide (5).
MATERIALS AND METHODSBacterial luciferase, isolated from Achromobacterfischeri, strain MAY (6), was purified-(7) and stored at -20°before use.Since it was prepared by a short procedure, there remained some impurities, especially some absorbance in the visible range. These were not large enough, however, to interfere with the observations. Its specific activity with FMNH2, determined at the time these experiments were done, was 4.6 X 10'3 quanta sec-' mg-1, with dodecanal at 220. Electrophoresis on acrylamide gels indicated a purity of about 80%. Bioluminescence was measured with a photometer (8) calibrated with the standard of Hastings and Weber (9).As already described for other enzymes (3), a medium appropriate for studies at low temperatures must be an innocuous organic solvent mixed with an aqueous system to depress the freezing point, and adjusted to suitable ionic environment and proton activity. For luciferase, a suitable medium in which the enzyme is active and not denatured at low temperatures is 50%/, ethylene glycol-lQ mM phosphate buffer (pH 7) (10). Under these conditions the actual "proton activity" (pH*) of the medium is 7.6 at +200 and 7.8 at -20° ( 11)
Absorbance and fluorescence spectra of bacterial cytochrome P-450cam and cytochrome P-450lin have been studied as a function of pressure. These pressure-induced spectral perturbations fall into two categories, which are interpreted as resulting from denaturation domains and are discussed in terms of protein structural dynamics. The results presented herein support a view that these two bacterial cytochromes have large structural differences and suggest a picture in which the gellike cortex of each protein may play an essential role in stability and function.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.