RESUMO
As demonstrated by selected examples from our laboratories, CE is a unique methodology for purity control of synthetic as well as natural tissue-isolated biopolymers, a prerequisite before reliable biotestings should be performed. A combination of rapid matrix-assisted laser desorption ionization mass and CE electrophoretic mobility determinations facilitates primary sequence determinations of enzymatic peptide digest mixtures often making costly Edman degradations unnecessary. The enormous separation efficiency and large variety of different possible separation modes in CE, allow detection of single components in complex mixtures which is demonstrated by the apolipoprotein A-I determination in human blood serum in this communication.
Assuntos
Bioquímica/instrumentação , Biopolímeros/análise , Eletroforese Capilar/estatística & dados numéricos , Laboratórios Hospitalares , Laboratórios , Algoritmos , Sequência de Aminoácidos , Apolipoproteínas/análise , Análise Química do Sangue , Eletroforese em Acetato de Celulose , Humanos , Dados de Sequência Molecular , Nefelometria e Turbidimetria , Fragmentos de Peptídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Timosina/análiseRESUMO
A combination of high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) spiking, Edman degradation, amino acid analysis and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) was applied for the analysis of the primary structure of beta-thymosins. CE has been used to resolve peaks which coelute on HPLC, as well as to help establish the identity of tryptic fragments in the peptide mapping experiments of the two highly homologous beta-thymosins.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Eletroforese Capilar/métodos , Proteínas dos Microfilamentos/análise , Compostos Organofosforados , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Timosina/análise , Ubiquitinas/análise , Sequência de Aminoácidos , Animais , Bovinos , Pulmão/metabolismo , Dados de Sequência Molecular , Peptídeos/análiseRESUMO
A method for the determination of apolipoprotein A-I, the major protein compartment of HDL, in human serum is described. Rapid and easy serum sample preparation, well separated Apo A-I peaks in the human serum electropherogram and good linearity of the peak area vs. concentration plot, covering the range of the clinically relevant Apo A-I serum contents, suggest the introduction of this method routinely in clinical laboratories.
Assuntos
Apolipoproteína A-I/sangue , Eletroforese Capilar/métodos , Humanos , Nefelometria e TurbidimetriaRESUMO
Polysaccharides, isolated from the lichen Cetraria islandica, have antimicrobial effectiveness. For pharmaceutical applications the two glucan components lichenan and isolichenan as well as the galactomannan component are of actual interest. Especially the a-glucan isolichenan ist used as an active ingredient in cough lozenges. The conditions for the extraction of the raw material, mainly pH and temperature, have a strong influence on the yield of lichenan, isolichenan, and galactomannan, and also on the amount of tannins in the extract. Target products and also by-products give higher extraction yields with increasing extraction temperatures. Hot water extraction with subsequent fractionation of the extracted polysaccharides by multiple freezing/thawing steps and water removal applying ethanol and ether permitted the isolation of the target polysaccharides in preparative quantities. Tannins were removed by reversed phase chromatography. IR and NMR spectroscopy were used for structural characterization of lichenan and isolichenan. After optimization of the hot water extraction process no significant lower extraction and fractionation yields have been obtained compared to the established tricky DMSO extraction procedure.
Assuntos
Líquens/química , Polissacarídeos/química , Cromatografia Líquida de Alta Pressão , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Polissacarídeos/isolamento & purificação , Solubilidade , Espectrofotometria Infravermelho , Taninos/química , Taninos/isolamento & purificaçãoRESUMO
Capillary electrophoresis (CE) analysis of human serum proteins and apolipoproteins is described and compared with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and cellulose acetate membrane electrophoresis. Under optimized CE conditions apolipoproteins I could be determined directly in the serum and quantitated with a linear correlation between peak area and concentration up to a concentration of 100 mg/dL.
Assuntos
Apolipoproteína A-II/sangue , Apolipoproteína A-I/sangue , Proteínas Sanguíneas/análise , Eletroforese Capilar , Eletroforese em Acetato de Celulose , Eletroforese em Gel de Poliacrilamida , Humanos , Lipoproteínas HDL/sangue , MasculinoRESUMO
Comparison of high performance liquid chromatograms (HPLC) with capillary electrophoresis shows the latter to be superior in many cases owing to rapid separation, high resolution and high sensitivity. This is demonstrated with two examples (i) the isolation of natural peptides from bovine tissue and (ii) characterization of a synthetic peptide mixture with the natural sequence (fragment) of the human immunodeficiency virus (HIV) transmembrane glycoprotein gp 41 env.
