Thin-layer microcalorimetric studies of oxygen and carbon monoxide binding to hemoglobin and myoglobin.
A thin-layer gas-solution microcalorimeter has been developed to study the binding reactions of gaseous ligands with ligand binding macromolecules. We have measured the enthalpy of binding oxygen and carbon monoxide to horse myoglobin, human hemoglobin A0 and sperm whale myoglobin in phosphate buffer at pH 7.6, with the enzyme reducing system of Hayashi. Reactions of human hemoglobin were also done under various buffer conditions in order to elucidate the Bohr effect. These binding reactions were found not to exhibit a detectable enthalpy change over the temperature range of 10 degrees C to 25 degrees C. The enzyme reducing system was shown to react with oxygen in a manner that releases a substantial amount of heat. This problem was corrected by using a minimum amount and by placing the buffer and enzyme system in the reference cell effectively cancelling the oxygen enzyme reaction heat as well as the heat of gas dissolution. It was also demonstrated that glucose-6-phosphate, one of the reducing system components, in 50 mM concentrations can influence the heat of binding oxygen and carbon monoxide to hemoglobin. This effect was shown to be absent in the myoglobins and also with hemoglobin at glucose-6-phosphate concentrations less than 5 mM.
Johnson
C R
CR
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309.
Gill
S J
SJ
Peters
K S
KS
D000818:Animals; D002151:Calorimetry / Q000295:instrumentation / Q000379:methods; D002248:Carbon Monoxide / Q000378:metabolism*; D006441:Hemoglobin A / Q000378:metabolism*; D006736:Horses; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D008832:Microchemistry / Q000295:instrumentation / Q000379:methods; D009211:Myoglobin / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D010108:Oxyhemoglobins / Q000378:metabolism*; D014907:Whales
D007202:Indicators and Reagents; D009211:Myoglobin; D010108:Oxyhemoglobins; D002248:Carbon Monoxide; D006441:Hemoglobin A; D010100:Oxygen