The Binding of Organic Ions by Proteins. Comparison of Bovine and Human Serum Albumins

Klotz, Irving M.; Burkhard, R. K.; Urquhart, Jean M.

doi:10.1021/j150493a016

Cited by 44 publications

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“…These studies indicated that mechanis~ns of adsorption of organic anions by serum albumins from different species are similar (7). However, significant differences in total binding capacity for anions, and in mode of interaction, have been reported lately between samples of human and bovine serum albumin a t pH's above 6.8 (8,9). Studies reported here confirm and extend these observations.…”

Section: Introductionsupporting

confidence: 79%

“…Studies reported here confirm and extend these observations. Iclotz, Burkhard, and Urquhart (8,9) observed that as pH increased, total binding capacity of human and bovine albumin for methyl orange and related dyes increased unexpectedly but the increment was much greater for human albumin. In addition, there was an exaltation of the spectrum of dye anions bound to human albumin while the extinction coefficient of those bound to bovine albumin decreased slightly.…”

Section: Introductionmentioning

confidence: 99%

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THE pH DEPENDENCE OF THE ADSORPTION ISOTHERM AND ABSORPTION SPECTRUM OF METHYL ORANGE BOUND TO HUMAN AND BOVINE SERUM ALBUMIN

Colvin¹

1953

Can. J. Chem.

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The difference between adsorptiori isotlierlns and absorption spectra of methyl orange bound to human and to bovine albumill at pH 6.8, 9.1, and 11.0 has been s t~~d i e d .Exaltation of the spectrum of methyl orange bound to hurnan albumin is not necessarily correlated with total binding capacity. However, above pH 6.8, heterogeneity of the binding. sites for methyl orange on human albumin increases so markedly that it is reflected in a n appreciable increase in extinction coefficient for the first three or four ailions bound. The exaltation is acconipanied by a n increased -AFo of binding. Increase in ionic strength diminishes exaltation, and denaturation of the protein destroys it. These effects were not observed for bovine albumin. Results are interpreted in terms of a limited reversible expansion of the human protein molecules, without unfoltling, due to intraniolecular, electrostatic repulsion between groups.

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Section: Introductionsupporting

confidence: 79%

Section: Introductionmentioning

confidence: 99%

THE pH DEPENDENCE OF THE ADSORPTION ISOTHERM AND ABSORPTION SPECTRUM OF METHYL ORANGE BOUND TO HUMAN AND BOVINE SERUM ALBUMIN

Colvin¹

1953

Can. J. Chem.

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“…This mechanism could account for the continued failure of all attempts to find an actual plateau on graphs for the number of ions of warfarin bound, or any other anion noncovalently bound to albumin, plotted against the free ion concentration (1). The increase in binding strength of an anion to albumin when the pH is raised above 7.4 also occurs with bilirubin (17), L-tryptophan (18), testosterone (19), thyroxine (20), penicillin (21), organic dyes (13), and sulfa drugs (12).…”

Section: Discussionmentioning

confidence: 99%

Interaction of the anticoagulant drug warfarin and its metabolites with human plasma albumin

O’Reilly¹

1969

J. Clin. Invest.

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A B S T R A C T The interaction of the anticoagulant drug warfarin and its metabolites with human plasma albumin was studied by equilibrium dialysis. A 20-fold variation of buffer ionic strength (0.017-0.340) caused no significant change in the warfarin association constant. But the binding strength rose significantly as the pH was increased from 6.0 to 9.0 and then declined at pH 10.0. The 6-, 7-, and 8-hydroxywarfarin metabolites showed a 7-to 23-fold reduction in binding strength at pH 10.0. These data indicate that the molecular basis of the interaction is nonelectrostatic and that the introduction of polar hydroxyl groups on the coumarin nucleus by metabolism reduces its hydrophobic binding surface. The interaction was markedly exothermic and showed a positive entropy (increased molecular disorder), which suggests cooperative hydrogen and hydrophobic bonding as the molecular basis for the binding of warfarin to albumin.The marked albumin binding and nonpolar character of warfarin explains the respective absence and presence of the unchanged drug in urine and plasma of warfarintreated patients, while the more polar character and lesser albumin binding of the metabolites probably determines their absence in plasma and presence in urine. The relatively marked binding to albumin of the 4'-hy- INTRODUCTIONIn previous studies of the interaction of the coumarin anticoagulant warfarin sodium and human plasma albumin, the drug was strongly bound to plasma albumin when analyzed by equilibrium dialysis (1). While the strength of the interaction was studied as a function of temperature, no experiments were performed at different levels of buffer pH and ionic strength. Such experiments were performed in the present study which together with the thermodynamic data allowed us to analyze the molecular basis of the warfarin-albumin interaction.Countercurrent distribution analysis of plasma of patients receiving clinical doses of warfarin shows essentially only the unchanged drug and similar analysis of urine reveals only hydroxylated metabolites of warfarin (2, 3). To determine if the absence of the metabolites in plasma and the absence of unchanged drug in the urine could be correlated with albumin binding, we also studied the four known metabolites by equilibrium dialysis. Based on these data, a hypothesis is presented for the pharmacodynamics of warfarin in man. METHODS Warfarin sodiumMaterial. The equilibrium dialysis technique, the crystalline human albumin and warfarin sodium, the preparation of the cellophane bags, the experimental techniques employed, the measurement of free and bound drug, and the determination of the standard free energy change were exactly as described previously (1). The low concentrations of warfarin sodium were varied over a 16-fold range (6-100 ,moles/ liter), which evaluates mainly the association constant of the first warfarin anion bound (k1). The concentration of The Journal of Clinical Investigation Volume 48 1969 193 albumin used in all experiments was 0.4% (57.9 /hM/liter). The e...

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“…Introduction pH-dependent serum binding has been shown for a number of acidic and basic drugs, such as methyl orange (Klotz et al, 1952), barbiturates (Goldbaum & Smith, 1954), theophylline (Vallner et al, 1979), warfarin (Wilting et al, 1980), fusidic acid propranolol and oxprenolol , imipramine (Kristensen & Gram, 1982), quinidine and prazosin (Br0rs et al, 1984), and pethidine (La Rosa et al, 1984). For several of these drugs, 30% or more decrease in free fraction is observed if precautions to avoid a pH increase in serum are not taken , Br0rs et al, 1983.…”

mentioning

confidence: 99%

pH lability in serum during equilibrium dialysis.

Brørs¹,

Jacobsen²

1985

Brit J Clinical Pharma

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Changes in pH were determined in previously frozen normal human serum during dialysis against sodium phosphate, Krebs Ringer phosphate or Krebs Ringer bicarbonate buffers of pH 7.4. Serum was either untreated (native) or adjusted to pH 7.4 before dialysis. pH in native serum was 7.7-7.9 before dialysis, showed a decrease after 1 h, and an increase after 3 h. pH-adjusted serum showed a continuous pH increase during dialysis. The increase in serum pH during dialysis was larger at 370 C than at 220 C, larger at low than at high buffer molarity, and larger in native than in pH-adjusted serum. The observed changes in serum pH during dialysis are associated with unacceptably large errors in unbound fraction in serum for a number of important drugs. Drug binding determination in serum by equilibrium dialysis should be performed with buffers providing appropriate and stable pH level.

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The Binding of Organic Ions by Proteins. Comparison of Bovine and Human Serum Albumins

Cited by 44 publications

References 1 publication

THE pH DEPENDENCE OF THE ADSORPTION ISOTHERM AND ABSORPTION SPECTRUM OF METHYL ORANGE BOUND TO HUMAN AND BOVINE SERUM ALBUMIN

THE pH DEPENDENCE OF THE ADSORPTION ISOTHERM AND ABSORPTION SPECTRUM OF METHYL ORANGE BOUND TO HUMAN AND BOVINE SERUM ALBUMIN

Interaction of the anticoagulant drug warfarin and its metabolites with human plasma albumin

pH lability in serum during equilibrium dialysis.

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