International Journal of Biological Macromolecules 50 (2012) 1341–1345 Contents lists available at SciVerse ScienceDirect International Journal of Biological Macromolecules jo u r n al hom epa ge: ww w.elsevier.com/locate/ijbiomac Concentration dependence of chaperone-like activities of -crystallin, B-crystallin and proline Svetlana G. Roman a,b,∗ , Natalia A. Chebotareva a , Boris I. Kurganov a a Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, Moscow 119071, Russia b Department of Physics, Moscow State University, Leninskie Gory, Moscow 119992, Russia a r t i c l e i n f o Article history: Received 5 December 2011 Received in revised form 16 March 2012 Accepted 22 March 2012 Available online xxx Keywords: -Crystallin B-Crystallin Glycogen phosphorylase b Proline Aggregation UV irradiation a b s t r a c t Chaperone-like activities of -crystallin, B-crystallin and proline were studied using a test system based on aggregation of UV-irradiated glycogen phosphorylase b (Phb) from rabbit skeletal muscle. The biphasic character of the dependence of the initial rate of aggregation (v agg ) of UV-irradiated Phb on the concen- tration of -crystallin or B-crystallin is indicative of the existence of two types of chaperone–protein substrate complexes differing significantly in affinity between the components of the complex. The dependence of v agg on the proline concentration is sigmoid (Hill coefficient is equal to 1.6) suggest- ing that the positive cooperative interactions between the proline molecules bound on the surface of the protein particles occur. When studying the combined suppressive action of -crystallin and proline on aggregation of UV-irradiated Phb, a slight antagonism between proline used at a fixed concentration (0.15 M) and -crystallin was observed. At higher concentration of proline (0.5 M) each chaperone acts independent of one another. © 2012 Elsevier B.V. All rights reserved. 1. Introduction The chaperone-like activities of -crystallin, one of the repre- sentatives of the family of small heat shock proteins [1–6], and chemical chaperone proline [7–13] were studied by numerous investigators using different aggregation test systems (thermal aggregation of proteins, aggregation accompanying protein refold- ing, aggregation induced by relatively low concentrations of guanidine hydrochloride or urea and others). The main drawback of all these test systems is that a chaperone being tested can affect the stages preceding the aggregation stage. For example, when using a test system for which heat-induced unfolding of a protein substrate, glycogen phosphorylase b (Phb) or glyceraldehyde-3- phoshate dehydrogenase from rabbit skeletal muscle, precedes the aggregation stage, we observed that -crystallin [14–17] and pro- line at relatively low concentrations [12] could reveal accelerating effect on the stage of unfolding of the protein molecule. This circum- stance hinders the quantitative estimation of the chaperone-like activity. To overcome the above-mentioned drawback of aggregation test systems, we proposed a new test system based on aggregation ∗ Corresponding author at: Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, Moscow 119071, Russia. Tel.: +7 495 9525641; fax: +7 495 9542732. E-mail address: svetabaj@gmail.com (S.G. Roman). of UV-irradiated protein substrate (see [18]). In this work we showed that UV irradiation of Phb resulted in denaturation and inactivation of the enzyme molecules. Denatured Phb molecules are assembled in clusters with the hydrodynamic radius of 10.4 nm which are relatively stable at 20 ◦ C, however reveal a tendency to further sticking at higher temperatures (for example, at 37 ◦ C). The test system of such a type allows detecting the effect of agents possessing chaperone-like activity exclusively on the aggregation stages. It is significant that in this case we obtain the true charac- teristics of the chaperone-like activity of the agents being tested without distortions caused by the effect of the agents under study on the stage preceding the aggregation stage (for example, on the stage of protein denaturation). Analysis of data on titration of a protein by a ligand gives infor- mation on the stoichiometry of the protein–ligand complex and the affinity of the ligand to the protein. When molar concentra- tions of the protein and ligand exceed significantly the value of the dissociation constant for the protein–ligand complex, the titra- tion data allow the stoichiometry of the complex to be determined. When molar concentration of the ligand (inhibitor) exceeds signif- icantly molar concentration of the protein (enzyme), the titration data (or the dependences of the enzyme activity on the inhibitor concentration) allow the affinity of the ligand to the protein to be determined. In the present work we compared the antiaggregation activities of -crystallin, B-crystallin and proline with UV-irradiated Phb as a protein substrate. Analysis of the concentration dependences of 0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2012.03.015