Effect of 2-Hydroxypropyl-b-cyclodextrin on Thermal Stability and Aggregation of Glycogen Phosphorylase b from Rabbit Skeletal Muscle Tatyana B. Eronina, 1 Natalia A. Chebotareva, 1 Sergey Yu. Kleymenov, 2 Svetlana G. Roman, 1,3 Valentina F. Makeeva, 1 Boris I. Kurganov 1 1 Department of Structural Biochemistry of Proteins, A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky Prospect 33, Moscow 119071, Russia 2 Koltsov’s Institute of Developmental Biology, Russian Academy of Sciences, Vavilova 26, Moscow 119334, Russia 3 Department of Physics, Moscow State University, Leninskie Gory, Moscow 119991, Russia Received 18 February 2010; revised 14 May 2010; accepted 5 June 2010 Published online 10 June 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.21508 This article was originally published online as an accepted preprint. The ‘‘Published Online’’date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com INTRODUCTION C yclodextrins are natural cyclic oligosaccharides con- taining several glucopyranose units linked through a- D-glucosyl units with an internal hydrophobic cavity and external hydrophilic edges. Many reports have indicated that protein stability against thermal and chemical denaturation and aggregation may be increased in the presence of cyclodextrins, which probably form complexes with accessible hydrophobic side chain of aminoacids. 1 For- mation of these complexes with proteins enhances their solu- bility in aqueous medium and diminishes protein aggregation inducing a decrease in the aggregate size. 2,3 Cyclodextrins can be useful stabilizing excipients in the preparation of spray- dried protein pharmaceuticals. 4 X-ray crystallography showed that b-cyclodextrin could bind to hepatic glycogen phospho- rylase in glycogen storage site with very little change in the ter- tiary structure acting as a poor inhibitor of the enzyme. 5 Cyclodextrins have relatively low solubility in water, so a great variety of cyclodextrin derivatives with improved aque- ous solubility have been developed, for example, 2-hydroxy- propyl-b-cyclodextrin (HP-b-CD). The 2-hydroxypropyl side groups located at the 02 positions widen the cavity en- trance at the secondary OH positions of the CD molecule (see Figure 1). These groups are spatially more spread out but dynamically more restricted due to the formation of a hydrogen bond network between the hydroxyl groups of the side chains and the glucose units. 6 HP-b-CD reduces aggre- gation of interleukin-2 during lyophilization, 7 stabilizes Effect of 2-Hydroxypropyl-b-cyclodextrin on Thermal Stability and Aggregation of Glycogen Phosphorylase b from Rabbit Skeletal Muscle Correspondence to: Tatyana B. Eronina; e-mail: eronina@inbi.ras.ru ABSTRACT: The study of the kinetics of thermal aggregation of glycogen phosphorylase b (Phb) from rabbit skeletal muscles by dynamic light scattering at 488C showed that 2-hydroxypropyl-b-cyclodextrin (HP-b-CD) accelerated the aggregation process and induced the formation of the larger protein aggregates. The reason of the accelerating effect of HP-b-CD is destabilization of the protein molecule under action of HP-b-CD. This conclusion was supported by the data on differential scanning calorimetry and the kinetic data on thermal inactivation of Phb. It is assumed that destabilization of the Phb molecule is due to preferential binding of HP-b-CD to intermediates of protein unfolding in comparison with the original native state. The conclusion regarding the ability of the native Phb for binding of HP-b-CD was substantiated by the data on the enzyme inhibition by HP-b-CD. # 2010 Wiley Periodicals, Inc. Biopolymers 93: 986–993, 2010. Keywords: glycogen phosphorylase b; aggregation; 2-hydroxypropyl-b-cyclodextrin; dynamic light scattering; differential scanning calorimetry Contract grant sponsor: Russian Foundation for Basic Research Contract grant number: 08-04-00666-a Contract grant sponsor: Presidium of the Russian Academy of Sciences (Program ‘‘Molecular and Cell Biology’’) V V C 2010 Wiley Periodicals, Inc. 986 Biopolymers Volume 93 / Number 11