Tryptophan identification

Although cysteine and tryptophan identification may be problematic, the data suggest that routine identification of these residues should be possible for the majority of facilities. 

Craig, A.G., Jimenez, E.C., Dykert, J., Nielsen, D.B., Gulyas, J., Abogadie, F.C., Porter, J., Rivier, J.E., Cruz, L.J., Oliveras, B.M., and McIntosh, J.M. (1997) A novel post-translational modification involving bromination of tryptophan identification of the residue, L-6-bromotryptophane, in peptides from Conus imperialis and Conus radiatus venom. J. Biol. Chem., 272, 4689-4698. 

Colabroy KL, TP Begley (2005) Tryptophan catabolism identification and characterization of a new degradative pathway. J Bacteriol 187 7866-7869. 

Griffiths, H.R, Lunec, J. and Blake, D.R. (1992). Oxygen radical-induced fluorescence in proteins identification of the fluorescent tryptophan metabolite N formyl kynurenine as a biological index of radical damage. Amino Acids 3, 183-194. 

Kleinberger-Doron, N. and Kanner, B. I. (1994) Identification of tryptophan residues critical for the function and targeting of the gamma-aminobutyric acid transporter (subtype A)../. Biol. Chem. 269, 3063-3067. 

Mazhul et alP have reported that long-lived luminescence could be detected in intact human erythrocytes and white blood cells at ambient temperature. They have shown by emission spectra and pH dependency that this emission arises from tryptophan. The emission was not singleexponential, suggesting that more than one population of tryptophan emitted. Identification of the emitting species has not yet been conclusively made, but the white blood cell protein content is about 10% actin, a protein known to phosphorescence.(91)... 

Chiara DC, Middleton RE, Cohen JB. 1998. Identification of tryptophan 55 as the primary site of [ H] nicotine photoincorporation in the y-subunit of the Torpedo nicotinic acetylcholine receptor. FEBS Lett 423 223-226. 

Three of these saccharides have to be N-acetylmuramic acid, and are either A, C, and E, or B, D, and F. The choice is easy, because the hydroxyl group on C-3 of carbohydrate C points toward the cleft and is hydrogen-bonded to the ring-nitrogen atom of L-tryptophan at position No. 63 of the protein core hence, there is no space for the lactoyl side-chain of N-acetylmuramic acid. Consequently, B, D, and F are N-acetylmuramic acid residues, and this identification is consistent with the low-resolution results. The lactoyl side-chains point out of the cleft, so that there is room for a peptide tail, such as occurs in the cross-linking of the cell walls of Micrococcus lysodeikti-cus.Ui... 

In conclusion, given a protein in which tryptophan is partially or wholly buried, quantitative estimates of quenching by different quenchers provide a further fingerprint that is specific to the particular conformation of a recombinant protein and which—combined with other fluorescence, circular dichroism, and gel electrophoresis data—forms the basis for specific identification and quality assessment. 

The more complex a-amino acids additionally show characteristic bands which aid in their identification. Thus in the spectrum of L-tryptophan (Fig. 3.37) the N—H stretching vibration, and the out-of-plane hydrogen wag deformation which appears at 742 cm-1 (four adjacent hydrogens), readily allow it to be distinguished from other a-amino acids. 

Ouwerkerk, P. B. F., Hallard, D., Verpoorte, R., and Memelink, J., Identification of UV-B light-responsive regions in the promoter of the tryptophan decarboxylase gene from Catharanthus roseus, Plant Mol. Biol., 41, 491, 1999. 

A 0.1% threshold for identification and isolation of impurities from all new molecular entities is under consideration by the International Conference on Harmonization as an international regulatory standard [4,5]. However, where there is evidence to suggest the presence or formation of toxic impurities, identification should be attempted. An example of this is the 1500 reports of Eosinophilia-Mylagia Syndrome and more than 30 deaths associated with one impurity present in L-tryptophan which were present at the 0.0089% level [6]. 

The identification of the radical in cytochromec peroxidase proved more elusive, partly because the EPR spectrum contains no well-resolved proton couplings. However, it was possible to identify this radical as being tryptophan-based by the effect of deuterated tryptophan on the couplings observed in the ENDOR spectra [27], No effect was observed when deuterated methionine was used. 

That chlorination occurs early in the biosynthesis of rebeccamycin is shown by the identification of two genes in the biosynthetic duster and their ability to chlorinate tryptophan at C-7 as shown in Scheme 19.5 [117]. 

The positive identification of W5 (82%) was a remarkable improvement over the previous study, ABRF-93SEQ, in which W2 and W7, also both early in the sequence, were positively identified 49% and 46%, respectively. Tryptophan 23 (W23) was positively identified (45%) nearly as well as the early tryptophan residues m ABRF-93SEQ. Positive cysteine identification (Cjo (53%) and C20 (37%)) improved over the 1993 study, where C5 was positively identified at 20%. 

In a few cases, alkaline hydrolysis has proved applicable to special problems. Tryptophan is not destroyed in alkali, and analysis of alkaline hydrolyzates forms the basis of one method for quantitative determination of this amino acid (e.g., Dreze, 1960). Despite the fact that tryptophan-containing peptides should be more stable in alkali than acid, partial alkaline hydrolysis has not been employed for identification of this type of peptide. Amino acids often can be regenerated by alkaline hydrolysis from derivatives obtained by the amino-terminal end-group methods. Dinitrophenyl amino acids and phenylthiohydantoin (Fraenkel-Conrat et al., 1955) as well as hydantoin (Stark and Smyth, 1963) derivatives of amino acids can be treated in this manner. 

The Association of Biomolecular Resource Facilities (ABRP) Protein Sequence Research Committee was established in 1988 in order to provide individual laboratories with a means of self-evaluation. Each year test samples have been distributed, enabling laboratories an opportunity to monitor their performance in areas such as sample handling, insuument operation/optimization, and data interpretation. In previous years these samples have focused on sensitivity of protein sequencing (1, 6), sample heterogeneity (2, 8), protein-bound peptides on PVDF membrane or in solution (3, 4), post-translational modifications (5), identification of cysteine and tryptophan (7), and length of sequence assignment (8). 

---