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

Abstract

The limits of cryofixation were probed by investigating metmyoglobin azide and carbonyl hemoglobin in ≈5wt% aqueuos 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 “hyperquenchin” either intheir 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(III) In the amide 1 spectral region of carbonyl hemoglobin (HbCO), a band at ≈ 1654cm −1 due to α-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 ≈1654 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 vittrified sample to 170 K. We surmise that HbCO is dehydrated by freeze-concentrated toa 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 perturbation of a protien than does quenching into a feeze-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. ≈ 170 K.