Sucrose cryoprotection

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. 

For cryostat sectioning, the tissue specimens are cryoprotected in 30% sucrose in 0.1 M phosphate buffer for 12 hr or until they sink to the bottom of the container. They are embedded in O.C.T compound (Miles, Elkhart, IN) and frozen in N-heptane cooled to the temperature of liquid nitrogen. Alternatively, if the antigens are resistant to paraffin embedding, the specimens can be dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin. 

Surimi is fish paste from deboned fish used to make simulated crab legs and other seafood. For preservation the paste is blended with cryoprotectants, such as sucrose, sorbitol and phosphates, and frozen. To make the final product, the frozen paste is thawed, blended with starch and extruded as a film onto a belt. The belt takes the film into an oven that heat-denatures the fish protein and cooks the starch. The film is then rolled to form striations, shaped, colored and cut. Depending on the required distribution, the product is frozen or refrigerated. Potato and tapioca starch were used in surimi products 400 years ago, since they provided a cohesive, elastic matrix consistent with seafood. Frozen distribution has made the use of highly-stabilized, moderately crosslinked tapioca starch popular, alone or with native tapioca starch. Modified waxy maize products are used, as is unmodified com starch, for increased cuttability. Kim188 reported that the gel strengthening ability of starch correlates with starch paste viscosity. 

The first published use of cryoprotectants for muscle proteins, found to be successful in commercial application, was a combination of sucrose (10%) and polyphosphate (0.2-0.5%) which... 

The cryoprotectant forms alay er of noncrystalline glass around the crystal to protect it from freeze shock. Simple freezing of the crystal results in the formation of ice in the interior of the crystal and renders it useless. A quick perusal of the literature shows PEG, glycerol, sucrose, and 2-methy 1-2,4-pentane diol (MPD) as the most popular cryopro-tectants. Oils, such as paraffin oil, have also been used successfully as cryoprotectants (12). 

More recently, there has been direct observation of protein structural perturbation in the frozen state using phosphorescence lifetime measurements [62]. Reductions in this parameter indicated that freezing perturbed the tertiary structure (at a protein concentration of 3-5 pM) of azurin, ribonuclease, alcohol dehydrogenase, alkaline phosphatase, glyceraldehyde 3-phosphate dehydrogenase, and LDH. The cryoprotectants sucrose and glycerol were tested and were found to inhibit the freezing-induced structural perturbations, with almost complete protection noted at a 1 M concentration. 

Finally, it is important to consider mechanistically how to explain the much greater potency of PEGs as cryoprotectants relative to other compounds such as sucrose. The data for one case, which are shown in Figure 9 and Table 1, illustrate... 

This argument does indeed support the contention that on a per-mole basis PEG is much more effective than sucrose at increasing protein chemical potential. And for cases where relatively high concentrations of PEG (e.g., >1% wt/vol) are needed to confer cryoprotection, the Timasheff mechanism may be applicable. However, it seems unlikely that a PEG concentration of 0.01% (wt/vol) would have a significant effect on the thermodynamics of the system. This is because the actual parameter of interest is the transfer free energy of the native versus denatured protein from water into cryoprotectant solution. The difference between the values for the two states determines the magnitude of the effect on the free energy... 

Figure 2. Effect of temperature on inactivation of thylakoids in the presence of NaCl. Washed thylakoids were suspended in a solution containing 100 mM sucrose and NaCl and were kept for 3 hours at 0°C, —6°C and —12°C. Freezing and thawing were fairly rapid and final temperatures were reached within less than 2 minutes. Sucrose served as cryoprotectant and was added to prevent freeze-inactivation of the membranes in the presence of low salt concentrations. After thawing, the activity of cyclic photophosphorylation was measured. Experimental conditions have been described previously (5, 20, 21).

Figure 3. Inactivation of thylakoids during freezing at various low temperatures as a function of time. Washed thylakoids were suspended in a solution containing 50 mM sucrose as a cryoprotectant and 20 mM sodium phenylpyru-vate as a cryotoxic solute. The suspensions were rapidly frozen and thawed. After thawing, photophosphorylation was determined. For experimental conditions, see notes in legend for Fig. 2...

Figure 5. Inactivation of thylakoids during freezing in the presence of chlorides of different divalent and monovalent metals. Washed thylakoids were suspended before freezing to —20°C in a solution containing 0.1 M sucrose as cryoprotectant ana various chlorides at concentrations indicated on the abscissa. After thawing, the activity of cyclic photophosphorylation was measured. For experimental conditions, see legend for Fig. 2.

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