Amino acid analysis advantages

There are several compounds that will react with amino acids to give coloured or fluorescent products and as a result can be used in qualitative or quantitative methods. Fluorimetric methods are gaining in popularity and offer some important advantages over absorption spectrophotometry for amino acid analysis. 

There are five categories of protein assay colorimetric assays, direct absorbance methods, fluorescence methods, amino acid analysis, and custom quantitation methods. A brief summary of the principles, advantages, and limitations of these methods follows. 

The separation power of CIEF often generates a high number of peaks even when relatively pure samples are analyzed. As already discussed, one of the advantages of CIEF is its potential micropreparative capabilities. Capillary IEF allows the collection of fractions that can be further analyzed by other methods. Some of the most widely used characterization tools include MS, peptide mapping, and amino acid analysis. 

The earliest approach to amino acid analysis involved postcolumn reaction. This scheme offers several advantages compared to precolumn reaction. First, it simplifies the sample preparation necessary. Often, precolumn derivatizations require sample cleanup steps to eliminate sample... 

The potential advantages of selective nitration of tyrosyl residues in native proteins are numerous. The reaction is performed under mild conditions, giving rise to a 3-nitrotyrosyl derivative (pK 7), which in the acid form absorbs intensely at 350 nm. Hence, the nitrotyrosine content may be readily determined spectrophotometrically, as well as by amino acid analysis ( 2.2.3). The absorption spectrum of 3-nitro-tyrosine is highly sensitive to solvent polarity and exhibits significant optical activity in the long wavelength absorption band. Consequently, nitrotyrosyl residues can be utilized as indicators of conformational change, or of interactions of proteins with other macromolecules or small molecules (e.g. Kirschner and Schachman 1973). Any perturbation in the pK of nitrotyrosyl residues is readily determined spectrophotometrically. 

For many applications it is important to know what the loading actually is (e.g. to compare activity of different enzymes immobilized to the same loading). Loading determination of an immobilized enzyme is often possible by methods such as quantitative amino acid analysis or active site titration. The latter has the advantage that only active enzyme is quantified. Typically, however, loading is determined indirectly during the immobilization process by measuring enzyme concentration in the aqueous solution before and after incubation with the carrier. The concentration of a pure enzyme in aqueous buffer can be determined from the absorption at 280 nm. The specific absorption can be calculated from the amino acid sequence of the enzyme. For less pure enzyme solutions, the total protein content can be determined with Bradford, BCA or other assays. 

Pre-column derivatization and subsequent chromatography of the amino acid derivatives potentially provide the highest sensitivity available for amino acid analysis (<10 15 molar). The Important advantage in this approach is that trace amino acid contaminants in the column eluents do not interfere as in the post-column systems. Only amino acids in the sample that have been derivatized are detected, and consequently the baseline is not influenced by possible contaminants contained in the eluents. 

There are three principal methods for cleaving peptide bonds to prepare hydrolysates suitable for amino acid analysis. Each has advantages and disadvantages, which have been described in great detail in other reviews and treatises (Light and Smith, 1963 Hill, 1965 Blackburn, 1968 Glazer et ai, 1975). Only the main features are discussed here. 

High-sensitivity amino acid analysis is usually done by precolumn derivatiza-tion (for a review, see Lottspeich and Henschen, 1982) followed by reverse-phase HPLC in an fully automated and dedicated instrument. The described combined o-phthaldialdehyde (OPA)/9-fluoromethoxycarbonyl (FMOC) derivatization of the amino acid has the advantage of being sensitive and, at the same time, reacting with all amino acids commonly present in proteins (Schuster, 1988). 

As mentioned in Section III, separation techniques for amino acids designed originally for PC are generally applicable for TLC amino acid studies on cellulose. Solvent systems for the TLC of amino acids generally employ mixtures of alcohols, acids or bases, and water. TLC is advantageous compared to PC for amino acid analysis in that it is faster and provides more compact spots, leading to better sensitivity and resolution of compounds. Experiment 1 provides a simple introduction to the TLC analysis of amino acid standards on silica gel. Experiment 2 provides a similar experience with cellulose and amino acid standards. Experiment 3 uses a reversed-phase layer to separate amino acids. For TLC exper-... 

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