Previously we have shown that human red blood cells (RBCs) undergo a sudden change from blocking to passing through a 1.3+/-0.2-microm micropipette when applying an aspiration pressure of 2.3 kPa at a critical transition temperature (Tc = 36.4+/-0.3 degrees C). Low-shear viscosity measurements suggested that changes in the molecular properties of hemoglobin might be responsible for this effect. To evaluate structural changes in hemoglobin at the critical temperature, we have used circular dichroism (CD) spectroscopy. The thermal denaturation curves of human hemoglobin A (HbA) and hemoglobin S (HbS) upon heating between 25 and 60 degrees C were non-linear and showed accelerated denaturation between 35 and 39 degrees C with a midpoint at 37.2+/-0.6 degrees C. The transition was reversible below 39 degrees C and independent of solution pH (pH 6.8-7.8). It was also independent of the oxygenation state of hemoglobin, since a sample that was extensively deoxygenated with N2 showed a similar transition by CD. These findings suggest that a structural change in hemoglobin may enable the cellular passage phenomenon as well as the temperature-dependent decrease in viscosity of RBC solutions.

Time-resolved and steady-state fluorescence, low-temperature phosphorescence, and optically detected magnetic resonance (ODMR) measurements have been made to resolve the luminescence contributions of the two intrinsic tryptophan residues in the subunits of trp aporepressor from Escherichia coli. Assignments of spectral information have been confirmed by use of the single-tryptophan mutants W19F and W99F. Solute fluorescence quenching studies show that both Trp19 and Trp99 are exposed to acrylamide and iodide, with Trp99 being the more exposed. Time-resolved and steady-state fluorescence measurements show Trp19 to have a bluer emission, a longer mean fluorescence decay time, a higher quantum yield, and essentially no independent rotational motion with respect to the protein. Trp99 is found to have a redder emission, a shorter mean fluorescence decay time, a lower quantum yield, and a significant degree of rotational freedom. Phosphorescence studies show a clear resolution of 0-0 vibronic transitions for each type of residue, with maxima at 407 and 415 nm that are assigned to Trp19 and Trp99, respectively. ODMR measurements show the zero-field splitting parameters to be quite characteristically different for each tryptophan residue. The existence of resonance energy transfer from Trp19 to Trp99, in the wild-type protein, is indicated by three types of data: comparison of the long-lived decay time (attributed to Trp19) in the absence (W99F) and presence (wild type) of the acceptor Trp99, comparison of the fluorescence quantum yield of the wild-type and mutant proteins, and deviations from the expected phosphorescence intensities for Trp19 and Trp99 in the absence of energy transfer.

The native lactose repressor from Escherichia coli (Lac Rep) and two single-point mutants, W220Y and W201Y, were investigated using low-temperature phosphorescence and optical detection of magnetic resonance (ODMR) spectroscopy. Emission from two tryptophan residues was evident in the phosphorescence spectrum of native Lac Rep at 77 K. Using the single-point mutants, the triplet-state properties of tryptophans 201 and 220 were obtained independently. Trp 220 was characterized as a partially solvent-exposed residue (0,0 band centered at 409.5 nm), while tryptophan 201 exhibited the properties of a buried residue (0,0 band centered at 413.5 nm). Both single-point mutant proteins experienced changes in tryptophan triplet-state properties as a result of binding either of two inducer sugars: isopropyl beta-D-thiogalactoside, a monosaccharide, or melibiose, a disaccharide. Putative singlet-singlet energy transfer from tryptophan 220 to tryptophan 201 was also investigated, but the quantitative results must be viewed with some caution.

Phosphorescence and optically detected magnetic resonance (ODMR) measurements have been carried out on the tryptophan (Trp) residues ofEscherichia coli Trp repressor protein (W Rep) and its two single Trp-containing mutants, W19F and W99F. The enhanced resolution afforded by the W19F and W99F mutants allowed us to characterize the triplet state of boundL-Trp corepressor using phosphorescence wavelengt-selected ORMR spectroscopy. We find that at 77 K the 0,0 band peak wavelength ofL-Trp is shifted from 405.5 nm in the aqueous solvent to ca. 410 nm when bound to the corepressor binding site. This red shift of the phosphorescence along with a corresponding increase in the zero-field splittingE value and narrowing of the ODMR linewidth characterize a binding site that is less polar, as well as more polarizable and homogeneous, than the aqueous solvent. This conclusion is in agreement with the X-ray crystallographic structure of the holorepressor protein that places the indole chromophore of the bound corepressor in a cleft in which it is sandwiched by the side chains of arginines 54 and 84.