Dataset Information

Self-association of human beta-galactocerebrosidase: Dependence on pH, salt, and surfactant.

ABSTRACT: Krabbe disease, also known as globoid cell leukodystrophy, is a rare genetic neurodegenerative disease caused by a deficiency of the galactocerebrosidase enzyme. To understand the association status of human beta-galactocerebrosidase (hGALC) in solution, we employed analytical ultracentrifugation (AUC). Our AUC results show that hGALC has a tendency for reversible self-association. Self-association decreases as the concentration of sodium chloride increases from 50 to 500 mM. This indicates that ionic interactions are involved in the association. The association is also dependent on pH, and high order oligomerization decreases as the pH increases from 4.5 to 7.5. Taken together, our results indicate that hGALC has the highest tendency for oligomerization at physiological ionic strength and pH (lysosomal lumen). This is the first report describing the self-associating property of hGALC in solution.

SUBMITTER: Lee E

PROVIDER: S-EPMC6927645 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Self-association of human beta-galactocerebrosidase: Dependence on pH, salt, and surfactant.

Lee Eunhee E Salamat-Miller Nazila N Stafford Walter F WF Taylor Katherine K

PloS one 20191223 12

Krabbe disease, also known as globoid cell leukodystrophy, is a rare genetic neurodegenerative disease caused by a deficiency of the galactocerebrosidase enzyme. To understand the association status of human beta-galactocerebrosidase (hGALC) in solution, we employed analytical ultracentrifugation (AUC). Our AUC results show that hGALC has a tendency for reversible self-association. Self-association decreases as the concentration of sodium chloride increases from 50 to 500 mM. This indicates that ...[more]

PMID: 31869371

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Project description:Although bovine beta-lactoglobulin assumes a monomeric native structure at pH 3 in the absence of salt, the addition of salts stabilizes the dimer. Thermodynamics of the monomer-dimer equilibrium dependent on the salt concentration were studied by sedimentation equilibrium. The addition of NaCl, KCl, or guanidine hydrochloride below 1 M stabilized the dimer in a similar manner. On the other hand, NaClO(4) was more effective than other salts by about 20-fold, suggesting that anion binding is responsible for the salt-induced dimer formation, as observed for acid-unfolded proteins. The addition of guanidine hydrochloride at 5 M dissociated the dimer into monomers because of the denaturation of protein structure. In the presence of either NaCl or NaClO(4), the dimerization constant decreased with an increase in temperature, indicating that the enthalpy change (DeltaH(D)) of dimer formation is negative. The heat effect of the dimer formation was directly measured with an isothermal titration calorimeter by titrating the monomeric beta-lactoglobulin at pH 3.0 with NaClO(4). The net heat effects after subtraction of the heat of salt dilution, corresponding to DeltaH(D), were negative, and were consistent with those obtained by the sedimentation equilibrium. From the dependence of dimerization constant on temperature measured by sedimentation equilibrium, we estimated the DeltaH(D) value at 20 degrees C and the heat capacity change (DeltaC(p)) of dimer formation. In both NaCl and NaClO(4), the obtained DeltaC(p) value was negative, indicating the dominant role of burial of the hydrophobic surfaces upon dimer formation. The observed DeltaC(p) values were consistent with the calculated value from the X-ray dimeric structure using a method of accessible surface area. These results indicated that monomer-dimer equilibrium of beta-lactoglobulin at pH 3 is determined by a subtle balance of hydrophobic and electrostatic effects, which are modulated by the addition of salts or by changes in temperature.

Project description:Spectrophotometric measurements of bilirubin-IX alpha in water and in aqueous/organic solvent mixtures at pH 10.0 as a function of bilirubin-IX alpha concentration (approx. 0.6--400 microM) are consistent with the formation of dimers (KD - 1.5 microM) in dilute (less than 10 microM) aqueous solution and further self-aggregation to multimers at higher concentrations. Added urea (to 10M) and increases in temperature (to 62 degrees C) obliterate the dimer-multimer transition at 10 microM, but added NaCl (to 0.30 M) promotes strong aggregation of dimers over a narrow concentration range, suggesting a 'micellization' phenomenon. Concentrations of dioxan or ethanol greater than 60% (v/v) in water were required to obtain the absorption spectrum of bilirubin-IX alpha monomers, suggesting that both hydrophobic and electrostatic (pi-orbital) interactions are involved in stabilizing the dimeric state in water. Micellar concentrations of sodium dodecyl sulphate induced spectrophotometric shifts in the dimer absorption spectrum of bilirubin-IX alpha consistent with progressive partitioning of bilirubin-IX alpha monomers into a relatively non-polar region of the micelles and allowed a deduction of the apparent critical micellar concentration that closely approximated the literature values. The pattern of bilirubin IX alpha association with bile salts is complex, since the absorption spectrum shifts hypsochromically below and bathochromically above the critical micellar concentration of the bile salts. Consistent with these observations, bilirubin IX alpha appears to bind to the polar face of bile salt monomers and to the polar perimeter of small bile salt micelles. At higher bile salt concentrations some-bilirubin-IX alpha monomers partition into the hydrophobic interior of the bile salt micelles. Our results suggest that under physiological conditions the natural conjugates of bilirubin-IX alpha may exhibit similar physical chemical properties in bile, in that dimers, highly aggregated multimers and bile salt-associated monomers may co-exist.

Dataset Information

Self-association of human beta-galactocerebrosidase: Dependence on pH, salt, and surfactant.

Publications

Self-association of human beta-galactocerebrosidase: Dependence on pH, salt, and surfactant.

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