Cryoprotective Effects of Prolyldipeptides and Prolyltripeptides on Lactate Dehydrogenase

ABSTRACT Proteins are not only essential nutrients, but also the active components of various biopharmaceuticals, including hormones, immune‐related molecules, and enzymes. The use of cryoprotectants is necessary to maintain the stability of protein drugs. Since most cryoprotectants require high concentrations to exhibit activity, there is a need for cryoprotectants that act at low concentrations. Although amino acids have been widely used as cryoprotectants, few studies have examined dipeptides and tripeptides as cryoprotective agents. This study identifies dipeptides and tripeptides with cryoprotective activity superior to that of amino acids. Screening experiments using lactate dehydrogenase (LDH), a cold‐sensitive model enzyme, showed that the prolyltripeptide Pro‐Leu‐Leu effectively inhibited LDH cryoinactivation, and this activity was approximately 100‐fold greater than that of proline, because the activity of 100 mM proline is roughly the same as that of 1 mM Pro‐Leu‐Leu. Pro‐Leu‐Leu also inhibited LDH denaturation induced by freezing and thawing (F/T) and prevented protein aggregation. Furthermore, Pro‐Leu‐Leu suppressed the cryoaggregation of human gamma globulin. Among prolyltripeptides, protective activity correlated weakly with hydrophobicity but did not correlate with their isoelectric point or net charge. Pro‐Leu‐Leu was among the most hydrophobic prolyltripeptides and exhibited the highest protective activity. These results suggest that Pro‐Leu‐Leu may limit the F/T‐induced expansion of hydrophobic regions exposed on the protein surface by covering hydrophobic patches via nonspecific interactions, thereby preventing protein denaturation and aggregation. Prolyltripeptides such as Pro‐Leu‐Leu may therefore be useful as efficient cryoprotectants for protein pharmaceuticals.