In situ spectroscopic quantification of protein-ice interactions

Abstract

FTIR and confocal Raman microspectroscopy were used to measure interactions between albumin and ice in situ during quasi-equilibrium freezing in dimethyl sulfoxide (DMSO) solutions. At temperatures of -4 and -6 °C, albumin was found to be preferentially excluded from the ice phase during near-equilibrium freezing. This behavior reversed at lower temperatures. Instead, DMSO was preferentially excluded from the ice phase, resulting in an albumin concentration in the freeze-concentrated liquid phase that was lower than predicted. It is hypothesized that this was caused by the albumin in the freeze-concentrated liquid getting adsorbed onto the ice surface or becoming entrapped in the ice phase. It was observed that, under certain freezing protocols, as much as 20% of the albumin in solutions with starting concentrations of 32-53 mg/mL may be adsorbed onto the ice interface or entrapped in the ice phase.

Figure 1

Binary DMSO and water phase diagram, adapted from the polynomial curve fit created by Kleinhans and Mazur to fit Rasmussen’s phase diagram.

Figure 2

Partially frozen solutions at −6 °C: (A) 6 wt % DMSO, 1:3 albumin/DMSO; (B) 8 wt % DMSO, 1:3 albumin/DMSO; (C) 10 wt % DMSO, 1:3 albumin/DMSO; (D) 12 wt % DMSO, 1:6 albumin/ DMSO.

Figure 3

FTIR and Raman albumin/DMSO ratio calibration: (A, B) Ratio of baseline-corrected areas for y and linear calibration curve. (C, D) Ratio of baseline-corrected areas for ζ and quadratic calibration curve.

Figure 4

(A–C) FTIR results: Change in albumin/DMSO ratio at −4, −6, and −8.5 °C. (D) Raman results: Change in albumin/DMSO at −17.5 °C.

Figure 5

Bulk freezing experiments: the albumin content in the FCL extracted from the samples after 30 h at −20 °C is plotted versus a measure of the effective surface area that could interact with the FCL. The γ values are normalized by the γ of the FCL extracted from the solution with the least amount of added ice (shown as 100%). The starting bulk solution is also included in the plot.