amino acids such as the reaction of tryptophan with 4-A method is described for determining the concen-(dimethylamino)benaldehyde (4). Biuret assays such as tration of membrane proteins and peptides in the presthe Lowery are based on the reaction of peptide bonds ence of non-amino-containing lipids. The assay is quanwith copper (5, 6), while dye-binding assays such as titative when used for purified proteins and peptides the Bradford depend on changes in absorbance when of known sequence and qualitative when sequences are chromophores interact noncovalently with proteins (7, unknown or samples contain contaminating proteins. 8). These methods are relative measures that compare In this method, proteins and peptides are hydrolyzed the spectroscopic results of a protein sample to those to amino acids followed by derivatization by fluoresobtained for a standard protein or proteins. Because camine and spectroscopic detection in a mixed solvent these assays are optimally selective for certain amino system. A liquid-phase acid hydrolysis separates lipid acids, differences between protein structures and from the sample and increases the sensitivity and accuamino acid sequences will influence their accuracy (9, racy of the assay. The aqueous-organic solvent, com-10). The last group of assays, hydrolysis of proteins, posed of 40% dimethylformamide, has two advantages. degrades proteins into their constituent amino acids First, it suppresses differences in fluorescence between (11, 12). Because hydrolysis methods analyze amino samples with and without residual hydrolyzed lipid, acids rather than whole proteins, accurate standards allowing direct comparison of samples and standards are easily obtained and not complicated by differing regardless of lipid content. Second, the solvent enprotein structures or sequence requirements. hances the fluorescence of amino acid derivatives.
For samples of membrane proteins or peptides, the While the fluorescence intensities of fluorescamine deremoval of lipid must be considered. Lipids interfere rivatives reach a maximum at approximately 40% diwith protein assays, requiring a separation step in the methylformamide, the emission maximum wavelengths continue to blue-shift at higher concentrations analysis of membrane samples. Previously reported of organic solvent. The selection of an acid hydrolysis methods for separation include extraction (13), protein mixture based on the fluorescence quenching by differprecipitation and/or adsorption onto filters (14-17), ent acid mixtures is also reported.