FLUORESCENCE CHANGES RESULTING FROM METHEMOGLOBIN AND FATTY‐ACID INTERACTION

Abstract

ABSTRACTInteraction between methemoglobin and saturated and unsaturated fatty acids was studied experimentally by fluorescence and absorption spectrum techniques, affinity chromatography and sedimentation coefficient measurements. Buffered solutions of linoleate, solubilized with Tween 20, when added to beef methemoglobin, and fresh rabbit oxyhemoglobin, caused a substantial increase in tryptophan fluorescence around 334 nm for an excitation frequency of 280 nm. At pH 7.8, at 25°C, 1 × 10−4 M linoleate added to 1.1 × 10−6M methemoglobin increased the quantum yield from 1.5% of that of the equivalent amount of tryptophan to 3.8%, and when added to the same concentration of oxyhemoglobin increased the quantum yield from 0.14% to 2.1%. Below pH.4 linoleate did not increase the fluorescence. The percentage increase in fluorescence is an exponential function of the number of carbon atoms in the saturated fatty acids. For linoleate the fluorescence dependence on linoleate concentration yielded Km= 1.8 × 10−4M. The increase in fluorescence was about three times greater from unsaturated than from saturated fatty acids. Methemoglobin in 5mM acetate buffer bound specifically to sepharose‐linoleate at pH 5 at 4°C and was eluted with 0.2M acetate buffer. The change in the absorption spectrum of the methemoglobin indicated a reduction of the methemoglobin upon the addition of linoleate. Band sedimentation in 0.5M KC1 of methemoglobin to which was added linoleate yielded s20,w= 2.58S compared to s2 0, w= 3.51S for the untreated methemoglobin. The results may be explained by a conformational change or dissociation of the methemoglobin tetramer.