Abstract
These experiments indicate that absorbance changes observed at the 425 nm isosbestic point of the Hb and HbCO following laser photolysis of HbCO provide a direct measure of the rates of quaternary conformational changes between rapidly reacting Hb (the immediate product of full photolysis) and slowly reacting normal deoxyhemoglobin. Hb, first observed by Gibson (Gibson, Q.H. (1959) Biochem. J. 71, 293-303), Has been interpreted as deoxyhemoglobin remaining in the liganded quaternary conformation following rapid removal of ligand by a light pulse. In borate buffers between pH 8.4 and 9.6 particularly simple pH-independent results were obtained which allowed the use of a Monod. Wyman, and Changeux model (Monod, J., Wyman, J., and Changeux, J (1965) J. Mol. Biol. 12, 88-118) to fit the data. In this case Hb is taken to be R state deoxyhemoglobin. Partial photolysis experiments at 425 nm show that the rate of the R - T conformational change at 20 degrees decreases by about a factor of 2 for each additional bound ligand. The rate of the ligand-free conformational change is found to be 920 +/- 60s(-1), 6400 +/- 600s(-1), and 15,700 +/- 700(-1) respectively at 3 degrees, 20 degrees, and 30 degrees. The previously uninterpreted effects of flash length and partial photolysis on the CO recombination kinetics can be explained in terms of the present model. Kinetic results obtained below pH 8 are found to be inconsistent with a two-state model. It appears that binding of inositol hexaphosphate produces a new rapidly reacting quaternary conformation of HbCO.
Highlights
These experiments indicate that absorbance changes observed at the 4‘25 nm isosbestic point of Hb and HbCO following laser photolysis of HbCO provide a direct measure of the rates of quaternary conformational changes between rapidly reacting Hb* and slowly reacting normal deoxyhemoglobin
Borate Buffer-In borate buffer the immediate product of full photolysis is taken to be deoxy material in the R quaternary state
No absorbance differences were seen between the immediate product of full photolysis of carboxy myoglobin, carboxy cyS;), or PSH chains and the deoxy derivatives
Summary
The effects reported here are distinct from those reported by Alpert et al [4]. Following laser photolysis of carboxyhemoglobin using a 30-ns light pulse from a Q-switched Nd glass laser, these authors observed absorbance changes which were complete in about 200 ns. These changes, which were observed for carboxy-~sH2 and -PSH chains and carboxymyoglobin, were interpreted as resulting from tertiary conformational changes of the protein following removal of bound carbon monoxide. On the longer time scale of these experiments, can be observed only with tetrameric hemoglobin
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