PubMed · 12584552
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Ronald L Poland. 2003. How to avoid kernicterus.. https://doi.org/10.1067/mpd.2003.72
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The interaction between human serum albumin (HSA) and the acetylcholinesterase inhibitor donepezil, has been studied by means of capillary electrophoresis frontal analysis (CE/FA) and circular dichroism. CE/FA enabled rapid and direct estimation of the quantity of free donepezil present at equilibrium with a physiological level of serum albumin (600 micromol L(-1)). Application of Scatchard analysis enabled estimation of the binding parameters of HSA towards donepezil, such as association constant and number of binding sites on one protein molecule. Furthermore, due to enantioseparation ability shown by HSA on donepezil in CE mode, displacement experiments were carried out using ketoprofen and warfarin as coadditives to the HSA based running buffer. The addition of these compounds reduced the enantioresolution of donepezil by HSA only when used at high concentration. These data were confirmed and corroborated by circular dichroism (CD) experiments. Using CD, bilirubin was also applied as a ligand specific to site III of HSA. The observed behaviour suggested that donepezil could be considered a ligand with independent binding to sites I and II; although site III is not the highest affinity site, indirect interaction (i.e. cooperative binding) can be assumed.
Here we report the use of IAsys biosensor technology for determining the binding parameters of low molecular weight compounds, such as warfarin and bilirubin, to surface immobilized human serum albumin. The protein was covalently immobilized on the surface of the biosensor cuvette, bearing a carboxymethyl dextran layer, through a condensing reaction between the carboxyl groups of the biosensor surface and epsilon-amine groups of protein lysine residues. This system detects and quantifies the changes in refractive index in the vicinity of the surface of the sensor chip to which the protein is immobilized. The changes in the refractive index are proportional to the change in the absorbed mass, thus the analysis allows the monitoring of the interaction process and the determination of the binding parameters. Optical biosensor analysis, most suited for studying protein/protein or protein/nucleic acid interactions, was sensitive enough to monitor the binding of low molecular weight compounds to human serum albumin and then suitable for a rapid screening of libraries of potential drugs when bioavailability is the research target.
Dilute aqueous and non-aqueous solutions of bilirubin were exposed to gamma radiation to examine the effects of ionizing radiation on the concentrations of a specimen of this nature. The ionising radiation emanated from a (137)Cs source, and was applied to 5.2 x 10(-2) mmol L(-1) solutions of the unconjugated specimen in 0.05 mol L(-1) aqueous NaOH and chloroform. Depletion of bilirubin after exposure was common to both solvents. Complete degradation was accomplished with doses in excess of 100 Gy. In the case of NaOH, it was found that the presence of molecular oxygen contributed more efficiently to the degradation process, than irradiation in air. When the experimental conditions were changed to nitrogen, the degradation process was suppressed. The sole by-product of merit originating from the NaOH work was the short-wavelength isomer of biliverdin, at 330 nm. In the case of chloroform, the exclusive product of interest was characterised as the long-wavelength isomer of biliverdin that absorbs in the broad region commencing from about 620 nm. The non-aqueous study was conducted in the presence and absence of molecular oxygen, with no significant changes in the results. Optimum production of the isomers in question occurred at a gamma dose of about 80 Gy. The general species of interest were monitored spectrophotometrically, and the results were treated mathematically to facilitate evaluation of the data. Our work represents the development of a facile gamma-ray method for the exclusive production of specific isomers of biliverdin, which are useful components in biosynthetic research.