Developmental Center-Driven Research Project)

FISK: M. Guo, Y. Cui,  J. McKee, J. Travierso, V. Alexander, A. Burger; 

UCD: Y. Yeh

The ability of antifreeze glycoprotein (AFGP) to modify the rate and shape of crystal growth has resulted in its use as food preservative and the cryostorage of cells in medicine and biotechnology. This project uses micro-Raman spectroscopy to probe the mechanism of how antifreeze glycoprotein adsorbs onto the surface of ice. By identifying the changes in the Raman spectral signature of AFGP during the protein’s adsorption process, we are beginning to reveal the mechanisms of thermal hysteresis and ice growth facet modification. In one set of experiments, we are using micro-Raman spectroscopy to determine the hydrogen bond related conformational changes of AFGP in a methanol aqueous solution as a function of temperature. These measurements reveal that the hydrogen bond is significant to the antifreeze absorption mechanism.

Lyophilized AFGP-8 was provided by the Yeh Laboratory at UCD. A high concentration aqueous solution was used for these temperature-dependent micro-Raman experiments. Methanol aqueous solution (concentration 80%) was used to verify the hydrogen bonding Raman shift. The spectra were collected by confocal micro-Raman system (LabRam from HORIBA Jobin Yvon using 11mW 632.8nm laser with 2mm diameter spot size). The Raman signature of AFPG-8 is shown to have a peak shift from 1065 cm-1 to 1047 cm-1 as the temperature is changed from 24 oC to -22 oC. This Raman shift is assigned to the AFGP (sugar ring)-H2COH function group with H-bond involved. The support experiments by methanol H3COH aqueous solution with H-bond involved (H3COH…HO-H) had confirmed this reasonable Raman shift from 1028 cm-1 to 1026 cm-1 as the temperature was varied from 26 oC to -26 oC. These measurements show the hydrogen bonding –H2C-OH…HO-H plays an important role in the binding process of AFGP molecules to ice with bonding involving disaccharide side chains of AFGP molecules such as the stachyose molecules.

As we continue to investigate the mechanism of action of antifreeze glycoproteins, we will also look at ways to validate and apply this new knowledge about the critical role of hydrogen-bonding in AFGP activity.