RESUMO
Protein entrapment and high-performance affinity chromatography were used with zonal elution to examine the changes in binding that occurred for site-specific probes and various sulfonylurea drugs with normal and glycated forms of human serum albumin (HSA). Samples of this protein in a soluble form were physically entrapped within porous silica particles by using glycogen-capped hydrazide-activated silica; these supports were then placed into 1.0 cm × 2.1 mm inner diameter columns. Initial zonal elution studies were performed using (R)-warfarin and L-tryptophan as probes for Sudlow sites I and II (i.e., the major drug binding sites of HSA), giving quantitative measures of binding affinities in good agreement with literature values. It was also found for solutes with multisite binding to the same proteins, such as many sulfonylurea drugs, that this method could be used to estimate the global affinity of the solute for the entrapped protein. This entrapment and zonal approach provided retention information with precisions of ±0.1-3.3% (± one standard deviation) and elution within 0.50-3.00 min for solutes with binding affinities of 1 × 10(4)-3 × 10(5) M(-1). Each entrapped-protein column was used for many binding studies, which decreased the cost and amount of protein needed per injection (e.g., the equivalent of only 125-145 pmol of immobilized HSA or glycated HSA per injection over 60 sample application cycles). This method can be adapted for use with other proteins and solutes and should be valuable in high-throughput screening or quantitative studies of drug-protein binding or related biointeractions.
Assuntos
Anticoagulantes/química , Cromatografia de Afinidade/métodos , Albumina Sérica/química , Varfarina/química , Sítios de Ligação , Produtos Finais de Glicação Avançada , Humanos , Cinética , Triptofano/química , Albumina Sérica GlicadaRESUMO
BACKGROUND: Novel, blue fluorescent solids referred to as Wild Plum compounds can camouflage skin imperfections when incorporated into cosmetic products. We evaluated the relationship between sun exposure and skin fluorescence and determined if the application of Wild Plum formulations could restore lost fluorescence without harming the skin. METHODS: The forehead skin of two groups of volunteers of mixed gender and age was examined for fluorescence and redness. In addition, subjects answered questions describing any adverse sensations they experienced after their skin was exposed to Wild Plum formulations for extended periods of time. RESULTS: Fluorescence measurements of both solar and non-solar skin indicated that repeated sun exposure causes a loss of skin fluorescence. Application of Wild Plum formulations caused an increase in skin fluorescence at all concentrations, restoring solar skin fluorescence to values well beyond that of non-solar skin. Photo analysis and interview questions indicated that these formulations did not cause any symptoms of irritancy. CONCLUSION: Wild Plum compounds have the ability to restore fluorescence of solar skin to a level significantly higher than that associated with non-solar skin. Skin appears more luminous and therefore more youthful. This fluorescence restoration is achieved at relatively low concentrations, without any harmful side effects.
Assuntos
Cosméticos/administração & dosagem , Corantes Fluorescentes/administração & dosagem , Envelhecimento da Pele/efeitos dos fármacos , Luz Solar/efeitos adversos , Raios Ultravioleta/efeitos adversos , Adulto , Idoso , Química Farmacêutica , Cor , Cosméticos/efeitos adversos , Emolientes/administração & dosagem , Emolientes/efeitos adversos , Feminino , Corantes Fluorescentes/efeitos adversos , Testa , Humanos , Masculino , Pessoa de Meia-Idade , Prunus , Higiene da Pele/métodos , Espectrometria de Fluorescência , Adulto JovemRESUMO
The binding of drugs with serum proteins and binding agents such as human serum albumin, α1-acid glycoprotein, and lipoproteins is an important process in determining the activity and fate of many pharmaceuticals in the body. A variety of techniques have been used to study drug interactions with serum proteins, but there is still a need for faster or better methods for such work. High-performance liquid chromatography (HPLC) is one tool that has been utilized in many formats for these types of measurements. Advantages of using HPLC for this application include its speed and precision, its ability to be automated, its good limits of detection, and its compatibility with a wide range of assay formats and detectors. This review will discuss various approaches in which HPLC can be employed for the study of drug-protein interactions. These techniques include the use of soluble proteins in zonal elution and frontal analysis methods or vacancy techniques such as the Hummel-Dreyer method. Zonal elution and frontal analysis methods that make use of immobilized proteins and high-performance affinity chromatography will also be presented. A variety of applications will be examined, ranging from the determination of free drug fractions to the measurement of the strength or rate of a drug-protein interaction. Newer developments that will be discussed include recent work in the creation of novel mathematical approaches for HPLC studies of drug-protein binding, the use of HPLC methods for the high-throughput screening of drug-protein binding, and the creation and use of affinity monoliths or affinity microcolumns for examining drug-protein systems.
RESUMO
Acetohexamide is a drug used to treat type II diabetes and is tightly bound to the protein human serum albumin (HSA) in the circulation. It has been proposed that the binding of some drugs with HSA can be affected by the non-enzymatic glycation of this protein. This study used high-performance affinity chromatography to examine the changes in acetohexamide-HSA binding that take place as the glycation of HSA is increased. It was found in frontal analysis experiments that the binding of acetohexamide to glycated HSA could be described by a two-site model involving both strong and weak affinity interactions. The average association equilibrium constant (K(a)) for the high affinity interactions was in the range of 1.2-2.0×10(5)M(-1) and increased in moving from normal HSA to HSA with glycation levels that might be found in advanced diabetes. It was found through competition studies that acetohexamide was binding at both Sudlow sites I and II on the glycated HSA. The K(a) for acetohexamide at Sudlow site I increased by 40% in going from normal HSA to minimally glycated HSA but then decreased back to near-normal values in going to more highly glycated HSA. At Sudlow site II, the K(a) for acetohexamide first decreased by about 40% and then increased in going from normal HSA to minimally glycated HSA and more highly glycated HSA. This information demonstrates the importance of conducting both frontal analysis and site-specific binding studies in examining the effects of glycation on the interactions of a drug with HSA.
Assuntos
Acetoexamida/química , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Albumina Sérica/química , Acetoexamida/metabolismo , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/metabolismo , Produtos Finais de Glicação Avançada , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/metabolismo , Ligação Proteica , Análise de Regressão , Albumina Sérica/metabolismo , Triptofano/química , Triptofano/metabolismo , Varfarina/química , Varfarina/metabolismo , Albumina Sérica GlicadaRESUMO
This study examined the preparation of high-capacity silica supports containing immobilized protein G. A maximum content of 39 mg protein G/g silica was obtained when using 100 A pore size silica, followed by 33 mg/g for 50 A silica and 9.3-24 mg/g for 300-4000 A silica. The surface coverage of protein G increased with pore size, with a maximum level of 0.037 micromol/m(2) being obtained for 4000 A silica. These supports gave comparable apparent activities (i.e., 30-47% binding to rabbit immunoglobulin G [IgG]), with the highest binding capacities (71-77 mg IgG/g silica) being obtained for 50-100 A silica.
Assuntos
Bioquímica/métodos , Proteínas Imobilizadas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Dióxido de Silício/metabolismo , Adsorção , Animais , Imunoglobulina G/metabolismo , Porosidade , Coelhos , Bases de Schiff/metabolismoRESUMO
A method is described for the entrapment of proteins in hydrazide-activated supports using oxidized glycogen as a capping agent. This approach is demonstrated using human serum albumin (HSA) as a model binding agent. After optimization of this method, a protein content of 43 (+/-1)mg of HSA/g support was obtained for porous silica. The entrapped HSA supports could retain a low-mass drug (S-warfarin) and had activities and equilibrium constants comparable to those for soluble HSA. It was also found that this approach could be used with other proteins and binding agents that had masses between 5.8 and 150kDa.