Figure 1.
Analytical size exclusion chromatography of Hbs with or without Hp.
A. Representative chromatograms of Hb, ααXLHb, and ββXLHb in the presence or absence of Hp. The absorbance was monitored at 405 nm for heme, and the peaks appearing between the elution time 13 and 19 minutes are Hb and Hp complexes. B. The percentage of Hp bound with Hb was determined and compared among ααXLHb, ββXLHb, and Hb based on baseline normalized area under curve for peaks within the boxed region of the eluting profiles (from Panel A). Statistical analysis was performed using a One-way analysis of variance with a Bonferroni’s Multiple comparison test to evaluate between group differences. Data are represented as mean areas +/− sem, significance was set at p<0.05. All analysis were performed using GraphPad Prism software.
Figure 2.
Native PAGE analysis of the Hb-Hp complexes.
The samples of Hb, ααXLHb, and ββXLHb with and without Hp were analyzed in 4–16% Native PAGE under the same condition. The positions of Hb dimer and tetramer, Hp dimer and polymers, and the Hb and Hp complex formation on gels were indicated relative to that of molecular weight markers.
Figure 3.
Rapid kinetics of Hp reaction with Hb measured by fluorescence emission change.
A. The time courses of Hp binding with Hb (15 µM) were fitted to exponential equations to obtain the pseudo-first-order rate constants for Hb, ααXLHb, and ββXLHb. B. The second order rate constants of Hp binding with native Hb (close circle) and ββXLHb (open circle) were derived from the Hb concentration dependence of the obtained rate constants.
Figure 4.
Stopped-flow kinetics of oxygen dissociation from Hbs in the presence and absence of Hp.
A. The time courses of oxygen dissociation from HbA (open cirlce), and the Hb and Hp complex (gray triangle) mixed with 1.5 mg/mL sodium dithionite (Na2S2O4) were plotted for comparison. B. The time courses of oxygen dissociation from ββXLHb (open circle), and the ββXLHb complex with Hp (gray triangle) were illustrated.
Table 1.
Ligand binding kinetic parameters of HbA and ββXLHb in the presence and absence of Hp.
Figure 5.
Stopped-flow kinetics of CO ligand association with HbA in the presence and absence of Hp.
A. Representative time course of CO (500 µM after mixing) binding with HbA was fitted to single exponential equation by non-linear least squares regression analysis. B. Representative time course of CO (250 µM after mixing) binding with the HbA and Hp complex was biphasic, and fitted to double exponentials to derive apparent association rate constants.
Figure 6.
Stopped-flow kinetics of CO association with ββXLHb in the presence and absence of Hp.
Representative time courses of CO (500 µM after mixing) binding with ββXLHb without (open circle) or with (open triangle) Hp were plotted for comparison. Both traces were fitted to single exponential equation by non-linear least squares regression analysis.
Figure 7.
Nitrite reaction with oxy HbA and ββXLHb in the presence and absence of Hp.
The spectral changes over time were measured in a spectrophotometer for the reaction of oxy HbA (30 µM) and nitrite (6 mM) in the absence (A) or the presence (B) of excess Hp. The kinetics of oxy HbA (C) and ββXLHb (D) reacting with freshly prepared nitrite as measured by rapid mixing, and the absorbance change was monitored at 577 nm. The time courses of the complex nitrite-induced Hb oxidation processes in the absence (open circle) and presence (open triangle) of Hp were illustrated, and the half times of the reaction under the same conditions were derived and listed in Table 2.
Table 2.
Hb redox reaction parameters in the presence and absence of Hp.
Figure 8.
Hydrogen peroxide-induced Ferryl/sulf ββXLHb formation in the presence and absence of Hp.
Ferryl Hb formation and stabilization measured as sulfHb concentrations in the reaction of metHb and H2O2. The close (•) and open (○) circles in the graph represent the sulfHb concentrations at reaction times of H2O2-induced metHb oxidation in the absence and presence of excess Hp, respectively. The inset shows typical spectral changes of resultant sulfHb at approximate 620 nm as a function of time. The approximate initial reaction rates were obtained and listed in Table 2.