Hydrogen bonding between sugar and protein is responsible for inhibition of dehydration-induced protein unfolding

The nature of the interaction responsible for the inhibition of protein unf olding and subsequent damage by sugars during dehydration is unclear. The r elationship between sample moisture content measured by coulometric Karl Fi scher titration and the apparent moisture content predicted by the area of the protein side chain carboxylate band at approximately 1580 cm(-1) in inf rared spectra of dried protein-sugar samples was examined. For samples in w hich a high level of native protein structure was retained in the dried sol id, the apparent moisture content predicted by the carboxylate band area wa s greater than the actual moisture content, indicating that protection resu lts from direct sugar-protein hydrogen bonding and not entrapment of water at the protein surface. Further, we show that the degree of structural prot ection conferred by sucrose and trehalose apparent in second derivative, am ide I infrared spectra, correlates with the extent of hydrogen bonding betw een sugar and protein. The failure of dextran to inhibit dehydration-induce d lysozyme unfolding is shown to result from the inability of the polymer t o hydrogen bond adequately to the protein. Therefore, formation of an amorp hous phase alone is not sufficient to maintain protein structure during deh ydration. Glucose hydrogen bonds to a high degree with dried lysozyme, but is incapable of inhibiting lyophilization-induced protein unfolding in the absence of an effective cryo-protectant. However, the addition of polyethyl ene glycol, which is known to protect proteins during freezing, but not dry ing, to glucose protected lysozyme structure during lyophilization. Togethe r, these results show that hydrogen bonding between carbohydrate and protei n is necessary to prevent dehydration-induced protein damage. However, hydr ogen bonding alone is not sufficient to protect proteins during lyophilizat ion in the absence of adequate freezing protection. (C) 1999 Academic Press .