Protein cryoprotective

Hincha, D.K., Heber, U. Schmitt, J.M. (1989). Freezing ruptures thylakoid membranes in leaves, and rupture can be prevented in vitro by cryoprotective proteins. Plant Physiology and Biochemistry 27, 795-801. 

Little is known concerning mechanisms by which these proteins prevent inactivation of thylakoids during freezing, but they somehow contribute to membrane stabilization. They act with some specificity, since cryoprotective proteins from spinach not only fail to protect red blood cells during freezing but are actually injurious. 

Sugar alcohols have also found appHcation in foods containing sugars. Sorbitol is an effective cryoprotectant in surimi, preventing denaturation of the muscle protein during fro2en storage. 

Most proteins are not sufficiently stable in aqueous solution to allow formulation as a sterile solution. Instead, the protein is freeze-dried and reconstituted before use. Development of a freeze-dried protein formulation often requires special attention to the details of the freezing process (potential pH shifts and ionic strength increase with freezing) as well as to potential loss of activity with drying. Formulation additives, such as sugars and polyhydroxy compounds, are often useful as cryoprotectants and lyoprotectants. Residual moisture may also be critical to the stability of the dried preparation [33],... 

Freeze-drying is a relatively gentle way of removing water from proteins in solution. However, this process can promote the inactivation of some protein types, and specific excipients (cryopro-tectants) are usually added to the product in order to minimize such inactivation. Commonly used cryoprotectants include carbohydrates (such as glucose and sucrose), proteins (such as HSA), and amino acids (such as lysine, arginine or glutamic acid). Alcohols/polyols have also found some application as cryoprotectants. 

Inclusion of a cryoprotectant in the formulation can stabilize the protein during the freezing and drying stages of lyophilization.17 Excipients frequently... 

Vemuri et al.17 looked at the effects of various cryoprotectants, freezing rates, and buffer systems on the shelf-life of lyophilized recombinant alphar antitrypsin (rAAT). Alpharantitrypsin (AAT) is labile in solution therefore, a more stable presentation was required. A competitive ELISA was used to measure total AAT in a sample. The AAT in the sample competed with HRP-labeled AAT for binding to the specific antibody. A stable formulation containing lactose as a cryoprotectant was found that maintained the protein s specific activity. 

III. X-Ray Studies at Subzero Temperatures Physical-Chemical Basis of Cryoprotection of Proteins in Solution and in the Crystalline... 

All these results are encouraging for investigators planning to use X-ray diffraction in mixed solvents at subzero temperatures and the rest of the present article will be devoted to a discussion of methods and preliminary results in this field. The methodology for cryoprotection of protein crystals, its physical-chemical basis, and the specific problems raised by the crystalline state, as well as the devices used to collect data at subzero temperatures, will be described. Limitations and perspectives of the procedure will be discussed critically. First attempts to determine the structure of productive enzyme-substrate intermediates through stop-action pictures will be described, as well as investigations showing that X-ray diffraction at selected normal and subzero temperatures can reveal protein structural dynamics. 

Work in solution is an absolute prerequisite for further studies of enzyme-substrate intermediates in the crystalline state. According to the Arrhenius relationship, k = A exp(—E IRT), which relates the rate constant k to the temperature, reactions normally occurring in the second to minute ranges might be sufficiently decreased in rate at subzero temperatures to permit intermediates to be stabilized, and occasionally purified by column chromatography if reactions are carried out in fluid solvent mixtures. Therefore, the first problem is to find a suitable cryoprotective solvent for the protein in question. 

Most protein crystals are not grown from naturally cryoprotecting solvents (even when an alcohol is the precipitant, its concentration is usually well below that required to prevent freezing below — 50°C) and therefore must be transferred into such solvents before cryoenzymologi-... 

Concluding this brief survey of the effects of cosolvents and temperatures on noncovalent binding forces between proteins, we may assume that while the dielectric constant may play a role in the cryoprotection of protein crystals, changes in interaction forces may confer protection or in some cases be responsible for crystal destruction. However, we must bear in mind that hydrogen bonds and salt links involved in the regions of contact between proteins will be strengthened and/or stabilized at low temperatures within certain limits of pan values, which should aid in the cryoprotection of protein crystals. 

Within organisms, organic sulfur is present predominantly as the amino acids cysteine and methionine, and the algal and bacterial osmolyte, dimethylsulfoniopropionate (DMSP). The latter also serves as an antioxidant and cryoprotectant. Small amounts of organosulfur are also present in some polysaccharides, lipids, vitamins, enzymes, and in the iron-sulfur protein ferrodoxin. Cell lysis and microbial degradation releases... 

Finally, Fig. 6.32 compares the internal dynamics of hydrated C-phycocyanin with that of the same system plus trehalose, a well known cryoprotecting disaccharide. Fig. 6.32 shows that the dynamics is slowed down by about 1.5 decades. The observation is interpreted as a slowing down of the protein dynamics due to the viscosity increase of the water shell by the added trehalose molecules. This finding is corrobated by the changes of the mean-squared-dis-... 

It is common for liquid nitrogen frozen protein crystals to acquire a patina of ice on the surface of the cryoprotectant. Diffraction of X-rays from even small ice crystals can mask reflections from the protein crystal. In addition, the presence of excessive amounts of ice can obscure the true position of the nylon loop, thereby resulting in the failure to place the crystal in the X-ray beam. It is, therefore, essential to remove ice crystals prior to diffraction analysis. 

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