A flow procedure to determine oxygen binding isotherms for low affinity and easily oxidized hemoglobins

Mekbib Astatke, William A. McGee, Lawrence J. Parkhurst

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

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 languageEnglish (US)
Pages (from-to)683-688
Number of pages6
JournalComparative Biochemistry and Physiology -- Part B: Biochemistry and
Volume101
Issue number4
DOIs
StatePublished - Apr 1992

ASJC Scopus subject areas

  • Biochemistry
  • Physiology
  • Molecular Biology

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