SPIN-LABELED HEMOGLOBIN DERIVATIVES IN SOLUTION POLYCRYSTALLINE SUSPENSIONS AND SINGLE CRYSTALS

The paramagnetic resonance spectra of the carbon monoxy, met, met azide, and met fluoride derivatives of horse hemoglobin spin labeled at the β93 cysteines have been observed in solution, polycrystalline suspensions, and single crystals. The β93 cysteines were alkylated with either N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide (I) or N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyI)iodoacetamide (II). At room temperature, the resonance spectra of all four derivatives labeled with either I or II show evidence of an equilibrium between two isomeric states of the spin label relative to the protein. The strong dependence of the resonance spectra upon buffer concentration and the ligand at the sixth coordination position of the iron atoms is mainly due to a displacement of this equilibrium. This dependence suggests small differences in the structures of the above derivatives that are evidently linked to the spin state of the iron atoms. However, in single crystals the principal hyperfine axis of each label has essentially the same set of orientations in all four derivatives, indicating that the protein conformation in the vicinity of the β93 cysteines must be very similar in all four. Differences in both the solution and crystal spectra decrease as the buffer concentration increases. Changes in the resonance spectra concomitant with crystallization can be detected but, again, the associated changes in protein structure are small. © 1969, American Chemical Society. All rights reserved.