Effects of drying methods and additives on structure and function of actin: mechanisms of dehydration-induced damage and its inhibition.
Limited stability impedes the development of industrial and pharmaceutical proteins. Dried formulations are theoretically more stable, but the drying process itself causes structural damage leading to loss of activity after rehydration. Lyophilization is the most common method used to dry proteins, but involves freezing and dehydration, which are both damaging to protein. We compared an air-drying method to freeze-drying to test the hypothesis that terminal dehydration is the critical stress leading to loss of activity. The secondary structure of air-dried and freeze-dried actin was analyzed by infrared spectroscopy and related to the level of activity recovered from the rehydrated samples. Actin dried by either method in the absence of stabilizers was highly unfolded and the capacity to polymerize was lost upon rehydration. The degree of unfolding was reduced by air-drying or freeze-drying actin with sucrose, and the level of activity recovered upon rehydration increased. The addition of dextran to sucrose improved the recovery of activity from freeze-dried, but not air-dried samples. Dextran alone failed to protect the structure and function of actin dried by either method, indicating that proteins are not protected from dehydration-induced damage by formation of a glassy matrix. In some cases, recovered activity did not correlate directly with the level of structural protection conferred by a particular additive. This result suggests that secondary structural protection during drying is a necessary but not sufficient condition for the recovery of activity from a dried protein after rehydration.
Allison
S D
SD
School of Pharmacy, University of Colorado Health Sciences Center, Denver, Colorado, 80262, USA.
Randolph
T W
TW
Manning
M C
MC
Middleton
K
K
Davis
A
A
Carpenter
J F
JF
D000199:Actins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D003890:Desiccation / Q000379:methods*; D003911:Dextrans / Q000494:pharmacology; D005079:Excipients / Q000494:pharmacology*; D005612:Freeze Drying / Q000009:adverse effects; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000187:drug effects; D011817:Rabbits; D013329:Structure-Activity Relationship; D013395:Sucrose / Q000494:pharmacology; D014867:Water / Q000737:chemistry
D000199:Actins; D003911:Dextrans; D005079:Excipients; D014867:Water; D013395:Sucrose