Limits of cryofixation as seen by Fourier transform infrared spectra of metmyoglobin azide and carbonyl hemoglobin in vitrified and freeze-concentrated aqueous solution.

The limits of cryofixation were probed by investigating metmyoglobin azide and carbonyl hemoglobin in approximately 5 wt% aqueous solution by Fourier transform infrared spectroscopy. Spectra of solutions cooled slowly and recorded in steps between 295 K and 190 K are compared with those obtained by "hyperquenching" either into their glassy states at 80 K, or into freeze-concentrated solution at 170 K. For metmyoglobin azide we conclude from an analysis of its covalently and ionically bound azide that it is impossible to freeze-in its high-spin/low-spin equilibrium even by hyperquenching, and that its vitrified state must correspond to a temperature T < 226 K for the Fe(II) site of the protein. In the amide I spectral region of carbonyl hemoglobin (HbCO), a band at approximately 1654 cm-1 due to alpha-helical structures is the dominant band in spectra recorded at ambient temperature and in the vitrified state, but in the spectrum of HbCO quenched at similar rates into a freeze-concentrated state, a band at approximately 1650 cm-1, tentatively assigned to unordered structures, becomes the dominant feature. This band is absent in the spectra of freeze-concentrated samples obtained by heating a vitrified sample to 170 K. We surmise that HbCO is dehydrated by freeze-concentration to a larger extent in solution quenched rapidly at 170 K than in a vitrified solution heated to 170 K, and that this dehydration is the primary cause for HbCO's perturbation. We conclude that freeze-concentration induced by heating a vitrified solution can cause less perturbations of a protein than does quenching into a freeze-concentrated state. Therefore it can be advantageous for the practice of freeze-etching to vitrify first a solution by "hyperquenching" and thereafter freeze-etch at e.g. approximately 170 K.

Alkali cation effect on carbonyl-hemoglobin's and -myoglobin's conformer populations when exposed to freeze-concentration of their phosphate-buffered aqueous solutions.

Freeze-concentrated aqueous phosphate-buffered (pH 6.8) solutions of carbonyl-hemoglobin (HbCO) and -myoglobin (MbCO) were investigated by Fourier-transform infrared spectroscopy for the effect of alkali cation on the population of conformers. When using sodium phosphates as buffer components, HbCO was transformed from conformer III (at approximately 1951 cm-1) which is the dominant form at ambient temperatures, into conformer IV (at buffer concentration at a given temperature. The conformational changes started slightly below the temperature where ice began to crystallize and the remaining solution became freeze-concentrated, and they were reversible for HbCO. For MbCO in 0.5 M sodium phosphate buffer solution, however, they were irreversible and MbCO denatured completely. When potassium phosphate salts were used for preparing the buffer at the same pH of 6.8, little or no transformation of conformer III into conformer IV was observed. The conformational changes induced by sodium salts are attributed to a decrease in pH and it is shown by infrared spectroscopy that during freeze concentration drastic changes in composition of the two buffer components H2PO4-/HPO(2)4- occur, the acid component increasing strongly relative to the base component. Supersaturation is also important because change from conformer III to IV requires a minimum concentration of sodium salts: whereas 0.1 M sodium phosphate buffer concentration shows a strong effect, 0.03 M concentration does not and therefore behaves like a potassium phosphate buffer.