Direct measurement of equilibrium constants for high-affinity hemoglobins.
The biological functions of heme proteins are linked to their rate and affinity constants for ligand binding. Kinetic experiments are commonly used to measure equilibrium constants for traditional hemoglobins comprised of pentacoordinate ligand binding sites and simple bimolecular reaction schemes. However, kinetic methods do not always yield reliable equilibrium constants with more complex hemoglobins for which reaction mechanisms are not clearly understood. Furthermore, even where reaction mechanisms are clearly understood, it is very difficult to directly measure equilibrium constants for oxygen and carbon monoxide binding to high-affinity (K(D) << 1 micro M) hemoglobins. This work presents a method for direct measurement of equilibrium constants for high-affinity hemoglobins that utilizes a competition for ligands between the "target" protein and an array of "scavenger" hemoglobins with known affinities. This method is described for oxygen and carbon monoxide binding to two hexacoordinate hemoglobins: rice nonsymbiotic hemoglobin and Synechocystis hemoglobin. Our results demonstrate that although these proteins have different mechanisms for ligand binding, their affinities for oxygen and carbon monoxide are similar. Their large affinity constants for oxygen, 285 and approximately 100 micro M(-1) respectively, indicate that they are not capable of facilitating oxygen transport.
Kundu
Suman
S
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.
Premer
Scott A
SA
Hoy
Julie A
JA
Trent
James T
JT
Hargrove
Mark S
MS
D001667:Binding, Competitive; D002245:Carbon Dioxide / Q000737:chemistry*; D003198:Computer Simulation; D000458:Cyanobacteria / Q000737:chemistry / Q000378:metabolism; D006454:Hemoglobins / Q000032:analysis / Q000737:chemistry* / Q000145:classification*; D007700:Kinetics; D008956:Models, Chemical; D012275:Oryza / Q000737:chemistry / Q000378:metabolism; D010100:Oxygen / Q000737:chemistry*; D040081:Protein Array Analysis / Q000379:methods*; D013053:Spectrophotometry / Q000379:methods*; D013329:Structure-Activity Relationship
D006454:Hemoglobins; D002245:Carbon Dioxide; D010100:Oxygen