<title>Heme-tryptophan relationships in hemoglobin explored by frequency-domain time-resolved fluorescence at 10-GHz resolution</title>

Abstract

We measured the time-resolved fluorescence spectroscopy of human and bovine oxy-and deoxyhemoglobins in either 0.03 M phosphate buffer or 0.03 M borate buffer between pH 6.5 and 9.2. A frequency resolved fluorometer was used with bandwidth up to 10 GHz. Excitation was at 294 nm, the emission was monitored through a broad band interference filter centered at 335 nm, coupled to a cut-off filter at 316 nm. In all cases, the best simulations were obtained with two discrete exponential decays, one near 30 ps, and the other of several hundred ps. In human hemoglobin, the longer component showed a substantial lengthening upon removal of oxygen. In bovine hemoglobin, the shorter component decreased upon deoxygenation. It was possible to infer that the shorter lifetimes originated from the average intrachain distances of tryptophans and hemes. Instead, the longer lifetimes were consistent with the interchain distances. However, the hemes at this longer distance would become the main acceptors of energy transfer only when the energy transfer at intrasubunit distance is inhibited. It is suggested that this is due to the presence of "disordered" heme in the system.

Spectroscopic innovations at the Laboratory for Fluorescence Dynamics

“…The instrument presented in Figure 1 has a flat frequency response to about 4 GHz offers high sensitivity and is immune from synthesizer phase noise [6]. Recently, it has been used to study the photophysics of hemoglobin intrinsic fluorescence [7]. Optical Multichannel Analyzer (OMA)…”