Amino acid analysis with selective

Four different amino acids have been selected for esterification to study the effect of R-group substituent of amino acid on rate and ease of esterification. The four acids are alanine, serine, aspartic acid and lysine. Their respective esters were prepared by reported methods to authenticate and compare with those prepared by our method. Alanine was esterified with ethanol to yield the ethyl ester, keeping -NH3+ group intact. This was also confirmed by acidity of final reaction mixture (pH- 3.2). There was about 50% conversion of alanine to its ethyl ester. Further work on ester formation, including qualitative and quantitative analysis, is in process. 

The critical problem of sample size and the limitations imposed thereby on the accuracy of elemental analyses, especially those for carbon and hydrogen, suggest the application of chromatographic techniques. Amino acid analysis may provide a superior method of assessing collagen loss with additional information regarding the selective decomposition processes, sensitive to local soil conditions. 

The Pirkle-type chiral stationary phases are quite stable and exhibit good chiral selectivities to a wide range of solute types. These CSPs are also popular for the separation of many drug enantiomers and for amino acid analysis. Primarily, direct chiral resolution of racemic compounds were achieved on these CSPs. However, in some cases, prederivatization of racemic compounds with achiral reagents is required. The applications of these phases are discussed considering re-acidic, re-basic, and re-acidic-basic types of CSP. These CSPs have also been found effective for the chiral resolution on a preparative scale. Generally, the normal phase mode was used for the chiral resolution on these phases. However, with the development of new and more stable phases, the reversed phase mode became popular. 

The versatility of this template was demonstrated with the synthesis of very short unusually helical polyalanine sequences stabilized by chaotrophic anions [35] and host systems for evaluation of the C-terminal helix capping propensities for nonpolar natural amino acids [36]. As model sequences WK4lnp2,LG-Hel-Ag-NH2 for the primary C-terminal amide and WK4Inp2 LG-Hel-A8-X-Inp-NH2 for candidate amino acids X were selected. In these sequences, Hel is the previously mentioned N-terminal helix template, Inp is 4-carboxypiperidine and L is tert-leucine. In the N-terminal region tryptophan (W) provides a UV reporter, four lysines (K4) are solubilizers, and Inp2 L a spacer element. The C-capping test region of these peptides is G-Hel-Ag-X-Inp, and its helicity is taken as proportional to [6)222, the per-residue ellipticity derived from CD analysis [37]. 

Figure 4. ExPASy Scores for tpis rabit When Data Were Submitted without or with Data from a Calibration Protein. Amino acid analysis data were submitted to the ExPASy site using the 16 residue Constellation 2 with (empty bars), and without (filled bars), known protein (calibrant) data furnished by the participants. The chart shows rank assigned to tpis rabit (SwissProt data base) for selected sites (a) Sites (n = 14) where accompanying data improved rank of tpis rabit. (b) Sites (n = 11) where including calibration data degraded the rank obtained for the query protein. For 16 sites (not shown), there was no change in rank of rabbit tpis with the inclusion of calibration data (see Table IV). Rank values above 10 are truncated.

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. 

If no derivatization takes place, detection is preferably accomplished by UV at a low wavelength (200-210 nm) in order to enhance detection sensitivity. However, detection selectivity is sacrificed at such low wavelengths. Electrochemical detection, when applied to the analysis of free amino acids, offers higher selectivity but suffers from a small linearity range. Furthermore, most amino acids (with the exception of tryptophan, tyrosine, and cysteine) are not intrinsically electrochemically active within the current useful potential range [5]. Lately, the development of the evaporative light-scattering detector (ELSD) offers an attractive alternative for the determination of nonderivatized amino acids (see Fig. 1). 

The identity of the defined peptides was confirmed by electrospray mass spectrometry [13,62] and the purity of defined peptides was higher than 80% as determined by HPLC. The amino acid composition of selected peptide libraries and of the sublibraries was determined by pool sequencing [14] (Fig. 11.1) and amino acid analysis. Deviations from equimolar representation of the amino acids in randomized sequence positions were found to be within the error limits of the analytical methods [12]. Electrospray mass spectrometry for pattern analysis of peptide libraries differing in the position of the defined amino acid was carried out with a triple quadrupole mass spectrometer (Fisons, Manchester, UK) (Fig. 11.2). 

Another example deals with amino acid analysis using immobilized specific microorganisms in combination with selective electrodes (35). Thus, glutamine could be analyzed by an electrode consisting of a potentiometric ammonia gas sensor and a layer of the bacterium Sarcina flava (American type culture collection 147) trapped in the volume between a NHo-permeable membrane on the surface of the electrode and a dialysis membrane in contact with the surrounding solution (Fig. 10). Using this electrode, steady state potentials were reached within 5 minutes. 

Finally, the important principle of subtractive analysis should be mentioned. Selective decomposition of the N-terminal residue is possible, for instance by deamination with nitrous acid or through oxidative deamination with ninhy-drin. Quantitative amino acid analysis before and after the reaction will show the absence of one amino acid in the hydrolysate of the treated sample. Obviously this is the residue with the free amino group hence it must have occupied the N-terminal position in the sequence. 

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