“…Linearization of the initial parts of the kinetic curves in the coordinates {( I − I
0 ) 0.5 ; t } (Fig. 2B) indicates that there exists a proportionality between the ( I − I
0 ) 0.5 value and time:where K
LS is a constant (such a designation for this constant was used in our previous works 35, 38, 39 ).Figure 2Kinetics of BSA aggregation registered by DLS (0.1 M Na-phosphate buffer, pH 7.0, 70 °C). ( A ) The dependences of the light scattering intensity ( I ) on time obtained at the following concentrations of BSA: 1.00 (1), 1.50 (2) and 1.75 mg∙mL −1 (3).…”
Section: Resultsmentioning
confidence: 99%
“…When interpreting the protective effect of these agents, we should know what kinetic regime of the aggregation process is realized 36, 39 . If we use test systems where the rate-limiting stage is that of protein unfolding (the order of aggregation with respect to protein is unity), the agents exerting some action on the stability of the native form of the target protein will eventually affect the initial rate of aggregation.…”
Chemical chaperones including arginine and its derivatives are widely used by biochemists working on the design of agents, which are able to efficiently suppress protein aggregation. To elucidate the mechanisms of anti-aggregation activity of chemical chaperones, methods based on registration of the increment in light scattering intensity must be supplemented with methods for direct detection of the portion of aggregated protein (γagg). For this purpose asymmetric flow field-flow fractionation was used in the present work. It was shown that heat-induced aggregation of bovine serum albumin (BSA) followed the kinetics of the reaction of the second order (0.1 M sodium phosphate buffer, pH 7.0, 70 °C). It was proposed to use R
h
vs γagg plots to characterize the aggregation pathway (R
h is the hydrodynamic radius of the protein aggregates, which was calculated from the dynamic light scattering data). The changes in the shape of R
h
vs γagg plots in the presence of arginine, arginine amide and arginine ethyl ester are indicative of the changes in the aggregation pathway of BSA aggregation. A conclusion has been made that larger aggregates are formed in the presence of arginine hydrochloride and its derivatives.
“…Linearization of the initial parts of the kinetic curves in the coordinates {( I − I
0 ) 0.5 ; t } (Fig. 2B) indicates that there exists a proportionality between the ( I − I
0 ) 0.5 value and time:where K
LS is a constant (such a designation for this constant was used in our previous works 35, 38, 39 ).Figure 2Kinetics of BSA aggregation registered by DLS (0.1 M Na-phosphate buffer, pH 7.0, 70 °C). ( A ) The dependences of the light scattering intensity ( I ) on time obtained at the following concentrations of BSA: 1.00 (1), 1.50 (2) and 1.75 mg∙mL −1 (3).…”
Section: Resultsmentioning
confidence: 99%
“…When interpreting the protective effect of these agents, we should know what kinetic regime of the aggregation process is realized 36, 39 . If we use test systems where the rate-limiting stage is that of protein unfolding (the order of aggregation with respect to protein is unity), the agents exerting some action on the stability of the native form of the target protein will eventually affect the initial rate of aggregation.…”
Chemical chaperones including arginine and its derivatives are widely used by biochemists working on the design of agents, which are able to efficiently suppress protein aggregation. To elucidate the mechanisms of anti-aggregation activity of chemical chaperones, methods based on registration of the increment in light scattering intensity must be supplemented with methods for direct detection of the portion of aggregated protein (γagg). For this purpose asymmetric flow field-flow fractionation was used in the present work. It was shown that heat-induced aggregation of bovine serum albumin (BSA) followed the kinetics of the reaction of the second order (0.1 M sodium phosphate buffer, pH 7.0, 70 °C). It was proposed to use R
h
vs γagg plots to characterize the aggregation pathway (R
h is the hydrodynamic radius of the protein aggregates, which was calculated from the dynamic light scattering data). The changes in the shape of R
h
vs γagg plots in the presence of arginine, arginine amide and arginine ethyl ester are indicative of the changes in the aggregation pathway of BSA aggregation. A conclusion has been made that larger aggregates are formed in the presence of arginine hydrochloride and its derivatives.
“…To calculate the n value, the series of kinetic curves of inactivation at various initial protein concentrations should be obtained. The order of inactivation with respect to the protein is a power coefficient in the expression connecting the initial rate of inactivation (v 0 ) and the initial protein concentration C 0 (Borzova et al 2015;Curto et al 2012;Delahaije et al 2015;Eronina et al 2016;Kurganov 2013aKurganov , b, 2015Kurganov , 2016Le Bon et al 1999;Roefs and De Kruif 1994;van Boekel 2009):…”
Section: Dissociative Mechanism For Irreversible Thermal Denaturationmentioning
Protein stability is a fundamental characteristic essential for understanding conformational transformations of the proteins in the cell. When using protein preparations in biotechnology and biomedicine, the problem of protein stability is of great importance. The kinetics of denaturation of oligomeric proteins may have characteristic properties determined by the quaternary structure. The kinetic schemes of denaturation can include the multiple stages of conformational transitions in the protein oligomer and stages of reversible dissociation of the oligomer. In this case, the shape of the kinetic curve of denaturation or the shape of the melting curve registered by differential scanning calorimetry can vary with varying the protein concentration. The experimental data illustrating dissociative mechanism for irreversible thermal denaturation of oligomeric proteins have been summarized in the present review. The use of test systems based on thermal aggregation of oligomeric proteins for screening of agents possessing anti-aggregation activity is discussed.
“…One of the important problems of modern biochemistry and molecular biology is shedding light on the mechanism of anti-aggregation activity of molecular chaperones (small heat-shock proteins) and low-molecular-weight osmolytes acting as chemical chaperons [ 1 ]. Test systems used for screening of agents possessing protective action are based on aggregation of client proteins induced by heating or by reduction of disulphide bonds in the protein molecule.…”
Section: Introductionmentioning
confidence: 99%
“…In general it is difficult to discriminate between the effects caused by the agents under study on the unfolding stage or the aggregation stage. To overcome this drawback, we have proposed to use aggregation of UV-irradiated proteins (glycogen phosphorylase b (Ph b ) and glyceraldehyde-3-phosphate dehydrogenase (GAPHD) from rabbit skeletal muscles) as test systems for studying anti-aggregation activity of chaperones [ 1 – 3 ]. In such test systems we are studying aggregation of UV-denatured proteins, and it is expected that the direct action of chaperones on the stage of aggregation can be estimated.…”
Different test systems are used to characterize the anti-aggregation efficiency of molecular chaperone proteins and of low-molecular-weight chemical chaperones. Test systems based on aggregation of UV-irradiated protein are of special interest because they allow studying the protective action of different agents at physiological temperatures. The kinetics of UV-irradiated glycogen phosphorylase b (UV-Phb) from rabbit skeletal muscle was studied at 37°C using dynamic light scattering in a wide range of protein concentrations. It has been shown that the order of aggregation with respect to the protein is equal to unity. A conclusion has been made that the rate-limiting stage of the overall process of aggregation is heat-induced structural reorganization of a UV-Phb molecule, which contains concealed damage.
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