A PULSED PHOTOLYSIS PROCEDURE FOR DETERMINING OXYGEN EQUILIBRIUM PARAMETERS OF LOW‐AFFINITY NONCOOPERATIVE HEMOGLOBINS

Mekbib Astatke, Lawrence J. Parkhurst

Research output: Contribution to journalArticlepeer-review

Abstract

Abstract— It is often very difficult to obtain precise values for oxygen equilibrium constants for low‐affinity nonco‐operative hemoglobins owing to the ease with which they are oxidized and denatured with even the most gentle stirring or agitation. We have developed a procedure that eliminates this problem by maintaining the hemoglobin as HbCO except during the brief time it is present as Hb, and HbO2 forms following photolysis. Oxygen is continuously removed by an enzyme system. The solution is photolyzed to 100% every2–3 min during the deoxygenation process to obtain maximum absorbance changes at the Hb‐HbCO isosbestic. These maximum absorbance changes at known times during the deoxygenation provide the necessary data for obtaining the oxygen equilibrium constant. These absorbance changes are used with enzyme kinetics equations to obtain calculated times. The simple equations, which neglect heme concentration, will be satisfactory for nearly all conditions, but for generality the complete equations are given. The variance minimizations are with respect to observed and calculated photolysis times. The results for native carp Hb over a range of temperatures are in excellent agreement with results obtained with considerably more material and greater difficulty by other methods.

Original languageEnglish (US)
Pages (from-to)964-971
Number of pages8
JournalPhotochemistry and Photobiology
Volume57
DOIs
StatePublished - May 1993

ASJC Scopus subject areas

  • Biochemistry
  • Physical and Theoretical Chemistry

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