Interactions of stabilizing additives with proteins during freeze-thawing and freeze-drying.
A wide variety of compounds, including sugars, polyols, amino acids and certain salts, are effective at minimizing protein denaturation during freeze-thawing. In this review we provide evidence that the mechanistic basis for this cryoprotection appears to be the same as that described by Timasheff for solute-induced stabilization in aqueous solution. Namely, the stabilizers are preferentially excluded from the surface of the protein, and this interaction makes it thermodynamically unfavourable for proteins to unfold. In contrast, carbohydrate-induced preservation of labile enzymes during freeze-drying is a fundamentally different process. Using Fourier transform intra-red spectroscopy we have found that hydrogen bonding between the carbohydrate and the dried protein is required for stabilization. Thus, it appears that certain carbohydrates protect dried enzymes because these solutes serve as water substitutes for the dried protein, by satisfying the hydrogen bonding requirement of polar groups on the protein's surface. Finally, we discuss some intriguing findings on the synergistic stabilization of proteins by mixtures of divalent cations and organic solutes, which are yet to be explained.
Carpenter
J F
JF
CryoLife, Inc., Marietta, GA 30067.
Arakawa
T
T
Crowe
J H
JH
D003451:Cryoprotective Agents; D004355:Drug Stability; D004798:Enzymes / Q000302:isolation & purification; D005612:Freeze Drying / Q000379:methods*; D011489:Protein Denaturation; D011506:Proteins / Q000302:isolation & purification*; D012996:Solutions; D014867:Water
D003451:Cryoprotective Agents; D004798:Enzymes; D011506:Proteins; D012996:Solutions; D014867:Water