A fluorescamine assay for membrane protein and peptide samples with non-amino-containing lipids.
A method is described for determining the concentration of membrane proteins and peptides in the presence of non-amino-containing lipids. The assay is quantitative when used for purified proteins and peptides of known sequence and qualitative when sequences are unknown or samples contain contaminating proteins. In this method, proteins and peptides are hydrolyzed to amino acids followed by derivatization by fluorescamine and spectroscopic detection in a mixed solvent system. A liquid-phase acid hydrolysis separates lipid from the sample and increases the sensitivity and accuracy of the assay. The aqueous-organic solvent, composed of 40% dimethylformamide, has two advantages. First, it suppresses differences in fluorescence between samples with and without residual hydrolyzed lipid, allowing direct comparison of samples and standards regardless of lipid content. Second, the solvent enhances the fluorescence of amino acid derivatives. While the fluorescence intensities of fluorescamine derivatives reach a maximum at approximately 40% dimethylformamide, the emission maximum wavelengths continue to blue-shift at higher concentrations of organic solvent. The selection of an acid hydrolysis mixture based on the fluorescence quenching by different acid mixtures is also reported.
Chung
L A
LA
Biochemistry Department, University of Virginia School of Medicine, Charlottesville 22908, USA. lac6m@virginia.edu
D000596:Amino Acids / Q000032:analysis; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004126:Dimethylformamide; D005450:Fluorescamine; D006851:Hydrochloric Acid; D006868:Hydrolysis; D008055:Lipids / Q000302:isolation & purification; D008565:Membrane Proteins / Q000032:analysis* / Q000302:isolation & purification; D010455:Peptides / Q000032:analysis* / Q000302:isolation & purification; D011422:Propionates; D012997:Solvents; D013050:Spectrometry, Fluorescence
D000596:Amino Acids; D008055:Lipids; D008565:Membrane Proteins; D010455:Peptides; D011422:Propionates; D012997:Solvents; D005450:Fluorescamine; D004126:Dimethylformamide; C029658:propionic acid; D006851:Hydrochloric Acid