Temperature-dependent conformation change in spin-labeled hemoglobin

Hemoglobin was spin labeled at beta-93(F9)-cysteine with N-oxyl-2,2,6,6-tetramethylpiperidinylmaleimide. The inward shift of the high-field hyperfine line (delta Hx) position in the ESR spectra of the spin label was measured as a function of temperature. One can except that an abrupt change in the microenvironment around the tightly bound spin label will be reflected in the function delta Hx(T) as a discontinuity (break point). This was shown for aquo-, azido-, nitro- and oxyhemoglobin derivatives. The presented results suggest that the microenvironment around the tightly bound spin label in those methemoglobin derivatives that exhibit the mixed-spin state of the heme iron is prone to an abrupt change above a certain ligand-specific temperature. The change in microenvironment of the spin label is probably due to a temperature-dependent change in the teritary structure of the protein.