1. 1. The determination of binding isotherms for low affinity hemoglobins is particularly difficult because of rapid oxidation. 2. 2. In carp Hb (pH 6 + 1HP, 25°C), one-quarter of the hemes are oxidized within 3 min, preventing the accurate determination of even P50 or X. 3. 3. We circumvent this problem by rapidly flowing HbO2, initially at pH 9 (X = 1.4 μM), against a low pH buffer to bring the system rapidly to equilibrium in the low affinity form. Diode-array spectrophotometry allows a complete spectrum to be obtained < 5 sec, after flow ceases, before significant oxidation has occurred. In tandem with the stopped flow apparatus is an oxygen electrode to measure O2 activity. 4. 4. At 22°C, the half-saturation oxygen activity (X) is 227 μM, and the Hill number is 0.91, for carp Hb (T state) implying significant differences in the O2 affinities of the alpha and beta chain hemes or, two different T states.
|Original language||English (US)|
|Number of pages||6|
|Journal||Comparative Biochemistry and Physiology -- Part B: Biochemistry and|
|State||Published - Apr 1992|
ASJC Scopus subject areas
- Molecular Biology