[Molecular bases of the fluorescence method in the determination of binding capacity of serum albumin]. / Molekuliarnye osnovy fliuorestsentnogo metoda opredeleniia sviazyvaiushchei emkosti al'bumina syvorotki krova.

A fluorescent method for assessment of albumin capacity to bind low-molecular metabolites, toxins, or drugs in blood serum, making use of fluorescent probe K-35, was recently suggested. The paper presents results of investigation of molecular basis of the method and of principles of interaction between fluorescent test molecules with albumin molecules in the blood serum. Molecules of fluorescent probe K-35 in blood serum plasma or serum are binding to albumin centers transporting low-molecular ligands (metabolites, toxins, drugs, etc.). Virtually the total intensity of K-35 fluorescence is due to the very molecules of the probe which are situated in these albumin centers. K-35 occupies two types of albumin centers, both of them equally contributing to total fluorescence intensity. Appearance of metabolites filling albumin centers and competing with K-35 probe results in reduction of the probe fluorescence. It is observed both in simulation experiments and in disease. It is possible that, besides the competitive mechanism, other mechanisms of blocking albumin centers in disease exist, to which K-35 is similarly sensitive. K-35 probe may be also used to measure effective albumin concentration.

[Fluorescent method of determining mass concentration of human serum albumin]. / Fliuorestsentnyi sposob opredeleniia massovoi kontsentratsii al'bumina syvorotki krovi cheloveka.

Fluorescent method for measuring mass (total) albumin concentration in human blood serum is suggested. Fluorophore K-35 previously suggested for measuring the effective concentration of albumin is recommended. Total albumin concentration is measured in acid pH range in the presence of nonionic detergent. Conditions under which the effects of factors impeding albumin assay virtually do not manifest were found. The resultant values of total albumin concentration coincide with the values determined by the bromocresol purple method.

[Anion-binding centers of human serum albumin during an N-F conformation transition and in isolated albumin and blood serum]. / Anion-sviazyvaiushchie tsentry syvorotochnogo al'bumina cheloveka pri konformatsionnom perekhod N---F v vydelennom al'bumine i v syvorotke krovi.

Fluorescent probe N-phenyl-1-amino-8-sulfonaphthalene (ANS) was used for studying pH-dependent structural N-F-transition in human serum albumin of two kinds: in commercial albumin and in natural blood serum. The kinetics of ANS fluorescence decay in albumin solutions was measured. There were found two types of the sites occupied by ANS in albumin under physiological conditions (pH 7.4). In the first binding site ANS fluorescence decay time was 16.6 +/- 0.3 nsec and it was not significantly changed at N-F transition (pH 4.0). In the second binding site the decay time was dependent on pH in commercial albumin and was not significantly changed in serum. In the second binding site there were individual differences of ANS decay time (4.3 +/- 0.6 nsec). The observed ANS fluorescence intensity enhancing (about 40-50%) in N-F transition may be explained by an increase of albumin binding sites capacity for ANS.

[Interaction of N-phenyl-1-amino-8-sulfonaphthalene (ANS) with albumin in blood in hyperbilirubinemia]. / Vzaimodestvie N-fenil-1-amino-8-sul'fonatalina (ANS) s al'buminom v syvorotke krovi pri giperbilirubinemii.

The fluorescent intensity of the N-phenyl-1-amino-8-sulfonaphthalene (ANS) probe significantly decreases in hyperbilirubinemic serum. A decrease of the albumin concentration and absorption of ANS fluorescence by bilirubin cannot explain such a considerable reduction of the probe fluorescence intensity. Measurements of the fluorescence decay kinetics has shown two types of sites occupied by ANS in albumin. ANS quantum yields in hyperbilirubinemic and normal serum are practically identical. The coupling parameters for ANS decrease, but the coupling constant increases under hyperbilirubinemia. As a result the coupling of organic anions with serum albumin significantly decreases if there is high anion concentration, and it does not decrease at low anion concentration. Bilirubin is not a main cause of a decrease of the albumin binding capacity.

[Fluorescent method of determination of serum albumin in hyperbilirubinemia]. / Fliuorestsentnyi metod opredeleniia al'bumina v syvorotke krovi pri giperbilirubinemii.

A procedure is developed for estimation of human blood serum albumin in hyperbilirubinemia using a fluorescent probe 9,10-dianiline-2-sulfoanthracene (K-33). Use of the probe N-phenyl-I-amino-8-sulfonaphthalene (ANS) led to underestimation of albumin in hyperbilirubinemia as bilirubin and other inhibitors prevented the ANS binding with blood serum albumin. Substitution of ANS for K-33 enabled to estimate the albumin concentration under conditions of the pathology accompanied by an increase of endogenous ligands content in blood serum. The rate of K-33 (15 mM) fluorescence in blood serum (diluted 400-fold, at pH 4.0) correlated with albumin concentration (r-0.955).

[The use of a fluorescent probe in assessing the binding ability of human serum albumin in liver insufficiency]. / Ispol'zovanie fliuorestsentnogo zonda v otsenke sviazyvaiushchei sposobnosti syvorotochnogo al'bumina cheloveka pri pechenochnoi nedostatochnosti.

Quenching of the fluorescence of the N-phenyl-1-amino-8-sulfonaphthalene (ANS) probe in the blood serum of icteric patients is due to not only bilirubin, but more so to other binding inhibitors characteristic of hepatic insufficiency of various etiology. The effects of these inhibitors on ANS binding with albumin may be essentially attenuated if the normal pH values be replaced by pH 4.0. The index, representing the ratio of the ANS fluorescence intensities in the blood serum at pH 7.4 and 4.0, characterizes the serum albumin binding capacity in hepatic insufficiency, and derivation of this index may be used as a clinical laboratory test.