Rapier H. McMenamy

ALBUMIN BINDING SITES

Publisher Summary This chapter presents albumin binding sites. The binding process to albumin has physiological significance in transport, modulation, inactivation of metabolite, and drug activities. It also acts as a protective device in binding and inactivation of potential toxic compounds to which the body is exposed. Forces available for the attachment of ligands to proteins are of several types, namely, electrostatic, fluctuating charges, hydrogen bonding, and apolar bonding. Some proteins bind anions, some do not. Compact proteins, supporting interiors of low dielectric constant, with positive charged groups, are potentially good anionic binders. This holds for albumin from almost all sources. Albumin binds most anions, independent of the hydrophobic character of the ligand side group, although if the latter is appreciable it clearly increases the affinity of attachment.

Microheterogeneity in albumin: A contaminant☆

Abstract Bovine Fraction V albumin in 3.3 m KCl has been subfractionated by lowering the the pH from 4.7 to 3.8. The association constants of skatole with nondefatted subfractions showed differences as large as twofold. Upon defatting, these differences disappeared. It was also found that variations in the pH solubility profiles of the subfractions were evident when albumin was not defatted, or when oleic acid or decanol were added to defatted albumin. Upon defatting the albumin, or omitting the additives (and allowing for variations in dimer content), all differences in solubility of the subfractions disappeared. No evidence was found for intrinsic heterogeneity in the defatted albumin. Details of a modified Goodman defatting procedure are given.

Association of indole analogues to defatted human serum albumin

Abstract The association constants of l -tryptophan, skatole, 3-indolepropionate, tryptamine and acetyl- l -tryptophan to defatted human serum albumin have been determined at two temperatures by dialysis equilibrium. These constants are generally different from those reported for nondefatted albumin. When thermodynamic values are assigned to the hydrophilic groups of the indolylethylene stem, Δ S u - d (the entropy change for the association after entropies of mixing and segment disorientation are subtracted) averaged 20e.u. less and Δ H ∘6 kcal. per mole less for the association to defatted albumin than to nondefatted albumin. For the indolylethylene stem Δ S u - d and Δ H ∘ were the same for defatted and nondefatted albumin. The pH of maximum binding of l -tryptophan shifted 1 unit higher after the albumin was defatted. With the defatted albumin an abrupt decrease in binding with pH for all compounds was found in the pH region 9.5–11.0, depending on the temperature. This decrease in binding was inversely related to the 3rd to 5th power of the H + concentration. Electrostatic effects were less in the associations to the defatted albumin than to the nondefatted albumin. Albumin reconstituted with fatty acid bound l -tryptophan similar to nondefatted albumin but not in all respects.

Thiocyanate binding with modified bovine plasma albumins

Abstract SCN − binding with guanidinated, acetamidinated, acetylated, carboxymethylated, β-dimethylaminopropionamidinated and cyanoethylated bovine plasma albumin has been studied. The binding at seven primary sites is not much affected in the modified proteins. Since the guanidinium groups are the only anionic binding groups unaffected by the modification reagents, this supports earlier evidence that the primary SCN − binding sites are located at arginyl residues. With respect to association at the secondary sites (presumably consisting predominantly of lysyl and imidazole groups) replacement of a hydrogen on the positively charged groups on the albumin by a bulkier radical is found to increase the binding constant of the site. Adducts which remove the positive charge on the binding site, on the other hand, appear to destroy the binding site.

Perifusion system for isolated cells

Abstract A system is described for perifusion of isolated hepatic cells which has a small interior space, rapidly responds to substrate changes, and requires no mechanical agitation of the cells. A 22 × 2-mm circular chamber is used with membranes (25 mm diameter, 8 μm pore size) at top and bottom. The solution enters the chamber at the bottom, perifuses the cells, and exits at the top. Gravity prevents cell clogging on the top membrane. Cells equivalent to 110–150 mg wet wt are perifused in a 0.7-ml chamber space. ATP levels, glucose and urea production, glucagon effects, and other measurements indicate excellent cell viability. O2 utilization is directly monitored. Examples of α-aminoisobutyrate and alanine uptake, and lactate and alanine metabolism in hepatocytes from 48-h fasted rats are given.

A method for determining amino acid concentrations and specific activities of amino acids and some other compounds in biological fluids

Abstract A method is described for obtaining plasma ultrafiltrates from which the concentrations of all amino acids, including tryptophan and ammonia, are obtained. A split-stream methodology is described for obtaining, in addition to the concentrations, the radioactivities of amino acids, glucose, and plasma water.

Binding of indole compounds with modified albumins: Characterization of the indole binding site

Abstract The binding of indole compounds (skatole, acetyl- l -tryptophan, l -tryptophan, indolepropionate) with modified bovine plasma albumin at the primary site has been studied. Guanidination and acetamidination of the e-amino groups (80 and 95% reacted, respectively) or carboxymethylation of the imidazole groups (90% reacted) reduces but does not destroy binding of skatole and acetyl- l -tryptophan. Guanidination of 30% of the e-amino groups increases the association constants of skatole, acetyl- l -tryptophan, and l -tryptophan. Acetylation of 25% of the e-amino groups by acetic anhydride has little effect on the association of skatole and indolepropionate but completely blocks association of l -tryptophan and acetyl- l -tryptophan. Other evidence indicates that the group responsible for blocking the binding of indole compounds with the α-amino and α-acetamidyl groups present (i.e. l -tryptophan and acetyl- l -tryptophan) is an e-amino residue of lysine in an apolar environment at the binding site. The indole ring part of the binding site remained intact (as evidenced by skatole binding) in all modifications of albumin except preparations extensively acetylated with acetic anhydride.

Indole-albumin association: a comparative study.

Abstract 1. 1. The binding of indole compounds ( d - and l -tryptophan, acetyl- d and l -tryptophan, skatole and indole-3-propionate) with albumin from eight mammals, three aves, one reptile and one amphibian has been studied. 2. 2. In every instance, except for the amphibian, the albumins are found to contain a unique site which binds the indole compounds with high affinity in a 1 : 1 complex. With mammals the l -indole analogues bind more strongly; with aves the d -indole analogues bind more strongly. Some differences in affinities of the various indole analogues also exist among animals of the same class. 3. 3. The specific binding trait is either lost during amphibian development or evolved shortly after amphibian development.

Plasma concentrations and tissue uptake of free amino acids in dogs in sepsis and starvation: effects of glucose infusion--some effects of low alimentation.

The plasma concentrations of substrates, together with transhepatic and transgut balances, have been studied in six control and eight septic awake fasted dogs. Four severely ill septic dogs (typically fluid in chest and/or abdomen, extensive peritonitis, respiratory difficulties) had high concentrations of threonine, glycine, tyrosine, lysine, histidine, tryptophan, and triglycerides (p less than or equal to 0.05). The other septic dogs (less severely ill) showed fewer and less pronounced alterations in the plasma substrates (aspartate and tryptophan were elevated, p less than or equal to 0.05). The infusion of glucose increased the concentration of glucose, lactate, and pyruvate and depressed the concentrations of most amino acids in both normal and septic dogs. Threonine, asparagine, glutamine, leucine, isoleucine, alpha-aminobutyrate, and tyrosine were significantly depressed in the severely ill septic dogs (p less than or equal to 0.05). In the normal dogs most amino acids were removed by the liver, with alanine accounting for approximately 40% of the total. Glutamine removal was negligible. In the septic dogs hepatic removal of amino acids was variable; livers of two severely ill septic dogs did not remove amino acids. In the control dogs glucose infusion (0.015--0.017 g/kg/min) tended to lower hepatic removal of amino acids. Hepatic dye removal in the septic dogs was always very poor. In the gut glutamine was removed and alanine, glutamate, glycine, and ammonia produced, but the overall sum of amino acid uptake was negligible in both the control and septic dogs. The ratio of tryptophan to the sum of valine, isoleucine, leucine, tyrosine, and phenylalanine concentrations was greatly elevated in all septic dogs in which it was measured. The free concentrations of amino acids in the liver, heart, and muscle tissues were grossly elevated in the low intravenous alimented septic state relative to the fasted normal state, whereas the tissue concentrative ability as measured by nonmetabolizable amino acids, alpha-aminoisobutyrate and cycloleucine, was not similarly increased. Sepsis clearly alters plasma and tissue concentrations, and in some instances hepatic uptake of amino acids.

A PROPOSED MECHANISM FOR THE BOHR EFFECT.

A change in the electrostatic potential between a macromolecule and the solution induces a pH change in the medium. Since mammalian hemoglobin is known to undergo structural alterations on addition of oxygen, the availability of a potential change adequate to explain the positive and negative Bohr effect is noted. This mechanism makes it unnecessary to assign the Bohr effect to changes in ionization of one or two specific groups on the hemoglobin molecule.