Date Published: May 6, 2016
Publisher: Public Library of Science
Author(s): Naoto Nishi, Takuya Miyamoto, Tomonori Waku, Naoki Tanaka, Yoshimichi Hagiwara, Xiaoming He.
The objective of this study is to enhance the inhibition of ice growth in the aqueous solution of a polypeptide, which is inspired by winter flounder antifreeze protein. We carried out measurements on unidirectional freezing of the polypeptide solution. The thickness of the solution was 0.02 mm, and the concentration of polypeptide was varied from 0 to 2 mg/mL. We captured successive microscopic images of ice/solution interfaces, and measured the interface velocity from the locations of tips of the pectinate interface in the images. We also simultaneously measured the temperature by using a small thermocouple. The ice/solution interface temperature was defined by the temperature at the tips. It was found that the interface temperature was decreased with an increasing concentration of polypeptide. To try varying the activity of the polypeptide, we preheated the polypeptide solution and cooled it before carrying out the measurements. Preheating for 1–5 hours was found to cause a further decrease in the interface temperature. Furthermore, wider regions of solution and ice with inclined interfaces in the pectinate interface structure were observed, compared with the case where the solution was not preheated. Thus, the ice growth inhibition was enhanced by this preheating. To investigate the reason for this enhancement, we measured the conformation and aggregates of polypeptide in the solution. We also measured the local concentration of polypeptide. It was found that the polypeptide aggregates became larger as a result of preheating, although the polypeptide conformation was unchanged. These large aggregates caused both adsorption to the interface and the wide regions of supercooled solution in the pectinate interface structure.
The inhibition of ice growth is an important issue in various fields, such as the maintenance of the quality of food texture in food preservation [1, 2]; the storage of cells, tissues and organs in hospitals [1, 3]; and cryosurgery . Antifreeze protein (AFP) and antifreeze glycoprotein (AFGP) have been investigated in relation to the inhibition of ice growth [1–3]. This is because the AF(G)P solutions have the following non-colligative properties: (a) the freezing point drop is not proportional to the AF(G)P concentration, (b) the melting point is retained, (c) the osmotic pressure does not significantly increase, and (d) specific pyramidal facets are observed on the surfaces of tiny ice crystals in AFP solutions in osmometers and on the surfaces of single-crystal ice-hemispheres which are grown from the end of a brass cold finger inserted into the surface of the solution [5, 6]. It should be noted that these freezing and melting points were defined to be respectively the temperatures at which a small seed crystal started to grow and shrink in the solution in an osmometer [7, 8].
We have carried out experiments on the gradual unidirectional freezing of the dilute solutions of polypeptide in a narrow gap between two cover glasses. This was inspired by winter flounder AFP. In addition, we measured the concentration of polypeptides, the absorbance of ultraviolet light and observed the transmitted electron beam and dynamic light scattering of the solutions being preheated. The main conclusions are as follows: