Quantification of anti-aggregation activity of chaperones: a test-system based on dithiothreitol-induced aggregation of bovine serum albumin.
The methodology for quantification of the anti-aggregation activity of protein and chemical chaperones has been elaborated. The applicability of this methodology was demonstrated using a test-system based on dithiothreitol-induced aggregation of bovine serum albumin at 45°C as an example. Methods for calculating the initial rate of bovine serum albumin aggregation (v agg) have been discussed. The comparison of the dependences of v agg on concentrations of intact and cross-linked α-crystallin allowed us to make a conclusion that a non-linear character of the dependence of v agg on concentration of intact α-crystallin was due to the dynamic mobility of the quaternary structure of α-crystallin and polydispersity of the α-crystallin-target protein complexes. To characterize the anti-aggregation activity of the chemical chaperones (arginine, arginine ethyl ester, arginine amide and proline), the semi-saturation concentration [L]0.5 was used. Among the chemical chaperones studied, arginine ethyl ester and arginine amide reveal the highest anti-aggregation activity ([L]0.5 = 53 and 58 mM, respectively).
Department of Molecular Organization of Biological Structures, Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia.
Vera A
VA
Borzova
Kira A
KA
Markossian
Dmitriy A
DA
Kara
Natalia A
NA
Chebotareva
Valentina F
VF
Makeeva
Nikolay B
NB
Poliansky
Konstantin O
KO
Muranov
Boris I
BI
Kurganov
D000818:Animals; D001120:Arginine / Q000494:pharmacology; D002417:Cattle; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D031521:Fractionation, Field Flow; D007700:Kinetics; D008027:Light; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D010316:Particle Size; D011392:Proline / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary; D058767:Protein Unfolding / Q000187:drug effects; D012031:Refractometry; D012542:Scattering, Radiation; D012710:Serum Albumin, Bovine / Q000737:chemistry* / Q000378:metabolism; D014461:Ultracentrifugation; D014783:Viscosity / Q000187:drug effects; D038201:alpha-Crystallins / Q000737:chemistry / Q000378:metabolism
D003432:Cross-Linking Reagents; D018832:Molecular Chaperones; D038201:alpha-Crystallins; D012710:Serum Albumin, Bovine; D001120:Arginine; D011392:Proline; D004229:Dithiothreitol