Figure 1.
Synchronous fluorescence spectra for the hemoglobin variants (1.0 µM) HbA, HbA2 and HbE with increasing temperature from 4°C-65°C.
(A-C) Represent the spectra for tryptophan (∆λ=80 nm) with main peak at around 280nm and auxiliary peak at 374nm and (D-F) represent those for the tyrosines (∆λ=20 nnm) with main peak at around 288nm and two auxiliary peaks at 348nm and 435nm. All spectra were taken at pH 7.0. The increasing intensities denote the solvent exposure of tyrosine and tryptophan residues. Auxilliary peaks in tryptophan indicate generation of other fluorophores or tyrosine derivatives.
Figure 2.
Thermal unfolding of HbA, HbE and HbA2 (10.0 µM) at pH 7.0 as recorded from (A) the change in the wavelength of Soret absorption maxima (415nm to 395nm) with increasing temperature, where bathochromic shift observed at temperature above 50°C, and (B) the change in absorbance in the Soret region (415nm) measured by uv-visible absorption spectroscopy.
The increased intensities beyond 60°C is due to aggregation of globin chains at higher temperature.
Figure 3.
Change in fluorescence intensity in the HbA, HbA2 and HbE (5.0 µM) as a function of temperature at pH 4.0.
The changes are reflected by (A) synchronous fluorescence for tryptophan emission, (B) synchronous fluorescence for tyrosine and (C) Change in synchronous fluorescence for tryptophan of HbA, HbA2 and HbE (1.0 µM) as a function of temperature at pH 2.5. Sharp decrease in intensity values in (A) and (B) indicate formation of unstable and insoluble aggregates which starts at a much lower temperature for HbE than HbA & HbA2 while at pH 2.5 all the variants become much more temperature sensitive as they are completely unfolded.
Figure 4.
Change in absorbance in HbA, HbA2 and HbE (10.0 µM) as a function of temperature at pH 2.5.
The slope of the lines indicate the order of thermal instability to be HbA>HbA2>HbE at pH 2.5. The error bars are Standard Errors of Mean (SEM) of 5 independent experiments which are of the order of the size of the symbols.
Figure 5.
Changes in intensities of (A) for dityrosine (Ex 315 nm, Em 400 nm) formed and (B) synchronous fluorescence of tyrosine in HbA, HbA2 and HbE (5.0 µM) as a function of temperature at pH 11.5.
The complete scan for the spectra related to (A) and (B) are presented in Figures S1 and S2 respectively.
Figure 6.
SDS-PAGE analysis of Hb variants (20 µg) incubated at pH 11.5 and at 40°C for 15 minutes.
Lane 1 shows the control, untreated Hb. Lane 2 shows the same after incubating in 5 mM H2O2 at pH 11.5 for 30 minutes and Lane 3 shows the same, as in Lane 2, after further incubation in 10 mM DTT for 15 minutes showing maximum yield of high molecular weight aggregates (band a and band b) in HbE than HbA and HbA2. Also appearance of band b in presence of H2O2 (Lane 2) and their disappearance in presence of DTT (Lane 3) indicate formation of intermolecular dityrosine.
Figure 7.
Thermal aggregation studies of HbA, HbA2 and HbE (45.0 µM) in presence and absence of 5 mM H2O2.
(A) Change in the intensity of 90° light scattering at 500 nm measured in a fluorescence spectrometer at pH 7 and (B) the same in the hydrodynamic radius as calculated by DLS measurements. Inset of (B) represents the homogeneity of the species under the light scattering experimental condition in terms of SOS values. Higher value indicates more heterogeneity in the solution in terms of radii of the species present. (C) Oxidative instability of HbA, HbA2 and HbE (45.0 µM) in presence of 5 mM H2O2 as reflected in the kinetics of aggregation, obtained from the change in hydrodynamic radius of the Hb variants with time. HbE shows a marked instability compared to the rest of HbA and HbA2 where aggregation starts within 180 second of monitoring.