Abstract
Reversible guanidine hydrochloride denaturation has been applied to obtain the first quantitative estimate of ligand-induced changes in hemoprotein conformational free energy. It is found that strong field (low spin) complexes, e.g. cyanometmyoglobin (MbCN) and azido metmyoglobin (MbN3), are 1.0 +/- 0.1 kcal/mol more stable than the high spin analogs aquometmyoglobin (MbH2O) and fluorometmyoglobin (MbF). This observed stability increment is essentially independent of the model chosen for data analysis. These results demonstrate the value of denaturation titration in measuring the stabilization of hemoprotein conformation by ligand binding. The denaturation of MbN3 appears complex. This complexity may be quantitatively accounted for by considering spin state equilibria. Applying this correction, MbCN and MbN3 have essentially the same stability in spite of steric differences in the two proteins. This result implies metal spin state is more important than ligand stereochemistry in determining the conformational free energy of myoglobin.
Highlights
Reversible guanidine hydrochloride denaturation has been applied to obtain the first quantitative estimate of ligand-induced changes in hemoprotein conformational free energy
It is found that strong field complexes, e.g. cyanometmyoglobin (MbCN) and azido metmyoglobin (MbNs), are 1.0 +- 0.1 kcal/mol more stable than the high spin analogs aquometmyoglobin (MbHzO) and fluorometmyoglobin (MbF). This observed stability increment is essentially independent of the model chosen for data analysis. These results demonstrate the value of denaturation titration in measuring the stabilization of hemoprotein conformation by ligand binding
The binding of ligands by hemoproteins has long been an area of active research [1]
Summary
Reversible guanidine hydrochloride denaturation has been applied to obtain the first quantitative estimate of ligand-induced changes in hemoprotein conformational free energy. It is found that strong field (low spin) complexes, e.g. cyanometmyoglobin (MbCN) and azido metmyoglobin (MbNs), are 1.0 +- 0.1 kcal/mol more stable than the high spin analogs aquometmyoglobin (MbHzO) and fluorometmyoglobin (MbF) This observed stability increment is essentially independent of the model chosen for data analysis. This complexity may be quantitatively accounted for by considering spin state equilibria Applying this correction, MbCN and MbN3 have essentially the same stability in spite of steric differences in the two proteins. MbCN and MbN3 have essentially the same stability in spite of steric differences in the two proteins This result implies metal spin state’is more important than ligand stereochemistry in determining the conformational free energy of myoglobin. I)100 stopped flow spectrometer or Cary 14 for slow reaction
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