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Studies of the oxygen binding equilibria in human hemoglobin
Abstract
Two new oxygen methods were developed for quantitating the binding of oxygen to Hb using enzymatic depletion of oxygen in solution. The first method used a computer controlled Cary 210 spectrophotometer to determine both oxygen activity and fractional saturation of Hb. The oxygen activity was calculated from the fractional saturation of myoglobin, determined from observations at the Hb/HbO$\sb2$ isosbestic wavelength. Fractional saturation of Hb was determined from absorbances at the Mb/MbO$\sb2$ isosbestic wavelength. The spectrophotometer cycled between these two wavelength during the deoxygenation. The deoxygenation of Hb was essentially complete in about 25 minutes. This procedure eliminated equilibration of Hb solutions with gas phase, and provided 250-500 independent data points per run. The second method also used enzymatic depletion of oxygen, where oxygen activity was measured by an oxygen electrode. Equilibration of the solution with a gas phase was eliminated. The change in Hb absorbance was monitored by a diode array spectrophotometer that collected 10,000 absorbance points over a 60 nm region for each run. The voltage from the oxygen electrode was smoothed by least-squares exponential splines and the undistorted electrode response was then obtained using iterative backward deconvolution to correct for the time constant of the oxygen electrode. While developing these methods, we found that fractional saturation for Hb was not directly proportional to the fractional absorbance change. The Adair constants were found to be wavelength dependent, and the apparent value of K$\sb4$, in particular, was dependent upon wavelength. The addition of three spectroscopic constants was needed to describe O$\sb2$ binding to the tetramer and allow K$\sb4$ to be within the bounds determined from kinetics. Oxygen equilibrium and kinetic measurements on cyano-met valency hybrids and on hemoglobin as a function of protein concentration showed little or no quaternary enhancement. We suggest that the common neglect of non-linear optical effects has given rise to the current controversy surrounding quaternary enhancement.
Subject Area
Biochemistry
Recommended Citation
Larsen, Todd M, "Studies of the oxygen binding equilibria in human hemoglobin" (1991). ETD collection for University of Nebraska-Lincoln. AAI9133297.
https://digitalcommons.unl.edu/dissertations/AAI9133297