Resolution of the lifetimes and correlation times of the intrinsic tryptophan fluorescence of human hemoglobin solutions using 2 GHz frequency-domain fluorometry.

E Bucci,H Malak,C Fronticelli,I Gryczynski,J R Lakowicz

doi:10.1016/s0021-9258(18)68591-6

E Bucci, H Malak + Show 3 more

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https://doi.org/10.1016/s0021-9258(18)68591-6

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Abstract

We used 2 GHz harmonic content frequency-domain fluorescence to measure the intensity and the anisotropy decays from the intrinsic tryptophan fluorescence from human hemoglobin (Hb). The tryptophan intensity decays are dominated by a short-lived component which accounts for 35-60% of the total steady state intensity. The decay time of this short component varies from 9 to 27 ps and this component is sensitive to the ligation state of Hb. Our error analyses indicate the uncertainty is about +/- 3 ps. The intensity decays also show two longer lived components near 0.7 and 8 ns, which are probably due either to impurities or to Hb molecules in conformations which do not permit energy transfer. The anisotropy decays indicate the tryptophan residues in Hb are highly mobile, with apparent correlation times near 55 ps.

Highlights

Ably short pulse width and thedetector must have picosecond resolution
Using this instrument we have found that the emission of tryptophan in extensively purified samples of hemoglobin was largely dominated by lifetimes in the picosecond region which were sensitive to ligation
Human hemoglobin was prepared from washed red cells obtained from fresh bloodsamples donated by the local bloodbank. They were hemolyzed in 0.005 M phosphate buffer at pH 7.0, and the stroma were eliminated by filtration through a 0.45 pm pellicon cassette

Summary

RESULTS

This emission is similar to that of tryptophan, except for the sharp peak near 325 nm. This argues against the presence of detectable amounts of apohemoglobin, at least in a form capable of binding hemin. We reasoned thatthe measured intensity would contain an increase proportion of the presumed intrinsic Hb fluorescence If this emission was dominated by scattered light, (with a decay time of 0) the anisotropy in the quenched samples may rise above 0.4 [20]. Acrylamide was not able to quench more than 50% of the emission, which is consistent

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DISCUSSION