Abstract
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).
Highlights
Folding of newly synthesized polypeptide chains can be accompanied by the formation of proteins prone to aggregation
Taking into account the data on bovine serum albumin (BSA) microheterogeneity caused by intramolecular disulfide interchange reactions [64], we can propose the following kinetic scheme of the aggregation process: Figure 21
DTT-induced aggregation of a-lactalbumin tends to decrease with increasing protein concentration and reaches a constant value at rather high concentrations of a-lactalbumin [18]
Summary
Folding of newly synthesized polypeptide chains can be accompanied by the formation of proteins prone to aggregation. SHsps, as a class of molecular chaperones, form a large family of ubiquitous proteins with molecular mass of subunit in the range 12–40 kDa, which are able to prevent protein aggregation. ACrystallin is a representative of a family of sHsps, exhibits chaperone-like properties, including the ability to prevent the precipitation of denatured proteins [1,2,3]. There is some evidence that the dissociated forms of sHsps are the chaperone-active species which interact with target proteins and are subsequently sequestered into high mass complexes [10,11,12,13]. The formation of complexes between dissociated forms of acrystallin and target substrates, muscle glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or glycogen phosphorylase b (Phb), at elevated temperatures has been demonstrated in our studies [19,20,21,22]
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