I. M. Vlasova

Investigation of denaturation of human serum albumin under action of cethyltrimethylammonium bromide by raman spectroscopy

The mechanism of denaturation of human serum albumin (HSA) under action of a cationic detergent—cethyltrimethylammonium bromide (CTAB) is investigated by Raman spectroscopy method. The percentage contents of α-helical segments in polypeptide chain of HSA at denaturation under action of different concentrations of CTAB at different values of pH is determined. It is shown, that more intensive denaturation of HSA under action of CTAB takes place at pH values, larger the isoelectric point of protein (pI 4.7).

Raman Spectroscopy in Investigations of Secondary Structure of Human Serum Albumin at Binding of Nanomarkers of Fluorescein Family

The influence of binding of nanomarkers of fluorescein family to HSA on secondary structure of this protein at different values of pH was investigated by Raman spectroscopy method. The greatest changes in secondary structure of HSA, consisting in decreasing of α-helix sites, at binding of fluorescein to HSA occur at pH 5–6. The greatest changes in secondary structure of HSA, consisting in decreasing of α-helix sites, at binding of eosin or erythrosin to HSA take place at values of pH, smaller 5. The differences in changes in secondary structure of HSA at binding of these three nanomarkers are explained by dependences of binding of nanomarkers to HSA on pH which determined by value of electronegativity of atoms of lateral radicals in structural formulas of nanomarkers and, therefore, by value of pK of their ionized groups.

Denaturation of human serum albumin under the action of cetyltrimethylammonium bromide according to fluorescence polarization data of protein

Denaturation of human serum albumin (HSA) under the action of cationic detergent cetyltrimethylammonium bromide (CTAB) is studied at different pH values by estimating the rotational diffusion of protein via fluorescence polarization. The degree of polarization of HSA tryptophan fluorescence, the rotational relaxation time, the rotational diffusion coefficient and the effective Einstein radius of the HSA molecules in solutions with different CTAB concentrations at different pH values are determined. The obtained rotational diffusion parameters of the HSA molecules show that under the action of CTAB, HSA denaturation has a one-stage character and proceeds more intensely and effectively at pH values higher than the pI value of protein (4.7).

Polarized Fluorescence in the Study of Rotational Diffusion of Human Albumin during Denaturation under the Action of SDS

Polarized tryptophan fluorescence of human serum albumin (HSA) was analyzed to determine the parameters of rotational diffusion (rotational relaxation time, rotational diffusion coefficient, and the effective Einstein radius) of HSA molecules during denaturation under the action of sodium dodecyl sulfate (SDS). Two stages of HSA denaturation under the action of SDS were shown: (1) loosening of protein globules and (2) unfolding of the amino-acid chain of the protein. HSA denaturation under the action of SDS is a two-stage process at pH values lower than the pI of HAS but passes through stage 1 only at pH values higher than the pI.

Raman Spectroscopy and Fluorescence Analysis in Investigation of the Protective Action of Ischemic Preconditioning at Ischemic Insult by Estimation of Damage of Low-Density Lipoprotein of Blood

OBJECTIVE AND BACKGROUND DATAnIn recent years medical researchers have sought a method of ischemic preconditioning, which is training an organ for prolonged total ischemia by inducing short periods of ischemia. In this work we detail research on this modality, conducted before ischemic insult in Wistar rats, in order to quantify the amount of damage done to low-density lipoprotein (LDL) in the blood. In previous research we used optical spectral methods to ascertain changes in LDL in the blood of animals after ischemic insult. The changes in LDL incurred by ischemic insult are explained by the free radical theory of oxidative stress.nnnMETHODSnWe used Raman spectroscopy and fluorescence analysis to determine if ischemic preconditioning performed before an ischemic insult renders protection against later ischemia, by analyzing reductions in damage done by free radicals to LDL.nnnRESULTS AND CONCLUSIONnThe adaptive effects of preconditioning were seen as the attenuation of the generation of free radicals by mitochondria of brain cells during total ischemia. Ischemic preconditioning also activates antioxidant systems, limiting the development of radical oxidative processes.

Rotational diffusion of fluorescein family markers in solutions of human serum albumin

Rotational diffusion of fluorescein family nanomarkers (initial fluorescein and its halogenated derivatives, eosin and erythrosine) in solutions of human serum albumin (HSA) was studied at various pH values. In solutions of HSA, the degree of fluorescence polarization, rotational relaxation time, and Einstein radius of nanomarkers are larger and the rotational diffusion coefficient of nanomarkers smaller than in solutions without the protein. An increase in the electronegativity of atoms in the structural formulas of nanomarkers increases the degree of polarization of their fluorescence, decreases the coefficient of their rotational diffusion, and increases rotational relaxation time and the effective Einstein radius.

Denaturation of bovine serum albumin initiated by sodium dodecyl sulfate as monitored via the intrinsic fluorescence of the protein

The tryptophan fluorescence of bovine serum albumin (BSA) is used to study the denaturation transitions in BSA under the influence of sodium dodecyl sulfate (SDS) at various pH values. The stepwise quenching of BSA tryptophan fluorescence and the gradual increase in the degree of anisotropy of BSA tryptophan fluorescence with increasing SDS concentration in solutions indicate the stepwise nature of denaturation: the first stage is a loosening of protein globules, whereas the second is a complete unfolding of the protein amino acid chain. At pH > pI of BSA, the denaturation BSA proceeds through both stages. At pH > pI of BSA, the denaturation BSA runs poorly and stops after the first stage. A more efficient BSA denaturation under the action of SDS occurs at pH < pI of BSA, with the efficiency of BSA denaturation under the influence of SDS decreasing with increasing pH.

Denaturation of bovine serum albumin under the action of cetyltrimethylammonium bromide, according to data from fluorescence analysis

The tryptophan fluorescence of bovine serum albumin (BSA) in solutions with different concentrations of cationic detergent cetyltrimethylammonium bromide (CTAB) at different pH is investigated, providing information on BSA denaturation under the action of CTAB. It is found that BSA denaturation under the action of CTAB at all of the investigated pH values (3.5–8.0) is a single-stage process, as determined by BSA tryptophan fluorescence quenching, by an increased degree of the BSA tryptophan fluorescence polarization, and by the values of the parameters for the rotational diffusion of BSA molecules in CTAB solutions. It is shown that the cationic detergent CTAB is more efficient for BSA denaturation at pH values higher than the BSA isoelectric point (4.9).

Denaturation of human serum albumin initiated by cetyltrimethylammonium bromide as monitored via the intrinsic fluorescence of the protein and fluorescence of an eosin probe

An analysis of the intrinsic fluorescence of the protein and the fluorescence of the eosin molecular probe in solutions of the protein was used to study the denaturation of human serum albumin (HSA) under the action of cetyltrimethylammonium bromide (CTAB), a cationic detergent, at various pH values. The denaturation of HSA under the influence of CTAB is a single-stage process over the entire pH range covered, pH (3.5–8.0). The maximum possible loosening of HSA globules in the presence of CTAB is reached at [CTAB] = 4 mmol/l. This detergent effectively denatures HSA at pH values higher than the pI of the protein (4.7).

The fluorescence characteristics and the molecular association of the Rose Bengal nanomarker in human serum albumin solutions

The decrease in the degree of molecular association of the Rose Bengal nanomarker in solutions with the addition of human serum albumin (HSA) has been revealed. It has been observed that in solutions with the addition of HSA the fluorescence quenching and the shifting of the fluorescence spectrum peaks of Rose Bengal to the red take place. It has been shown that the dependence of the effective binding constant of binding Rose Bengal to HSA steadily decreases with an increase in the pH value. It has been established that the values of the molecular association degree of Rose Bengal and the values of the effective constant of its binding to HSA depend on the magnitude of the electronegativity of the atoms in its structural formula, as well as on the pK values of its ionizable groups.