Tryptophan resolution

PTH amino acid analyzers used in this study were Applied Biosystems (59/78), Hewlett-Packard (16/78), Beckman (2/78), and Waters (1/78). The average age of these instruments was 5.2 2.6 years, and nearly all (76/78) were on-line with the sequencing system. Most users of Applied Biosystems sequencers reported using premix buffers (49/57), and several reported using additives to adjust the baseline (22/57) or to improve yields (18/57). The most common additive to Applied Biosystems solvent A was acetone (13/57), while others reported adding tryptophan (2/57) and TEA (2/57). Several facilities use dimethylphenylthiourea (DMPTU) to adjust the baseline rise (12/57) and isopropanol to improve tryptophan resolution in Applied Biosystems solvent B. 

Nakamura, M., Kiyohara, S., Saito, K. Sugita, K., and Sugo, T., High resolution of DL-tryptophan at high flow rates using a bovine serum albumin-multilayered porous hollow-fiber membrane, Anal. Chem., 71, 1323, 1999. 

Tryptophan (and also tryptophanol) undergoes a stereoselective cyclocondensation with racemic compound 249, in a very interesting process involving a kinetic resolution with epimerization of the tryptophan stereocenter and simultaneous desymmetrization of the two diastereotopic acetate chains <2005CC1327>, affording the enantiomeri-cally pure lactam 250. A subsequent treatment of the latter compound with trifluoroacetic acid led to the indolo[2,3- ]quinolizidine 251 through an intermediate acyliminium cation (Scheme 50) <20050L2817>. 

The elucidation of the intramolecular dynamics of tryptophan residues became possible due to anisotropy studies with nanosecond time resolution. Two approaches have been taken direct observation of the anisotropy kinetics on the nanosecond time scale using time-resolved(28) or frequency-domain fluorometry, and studies of steady-state anisotropy for xFvarying within wide ranges (lifetime-resolved anisotropy). The latter approach involves the application of collisional quenchers, oxygen(29,71) or acrylamide.(30) The shortening of xF by the quencher decreases the mean time available for rotations of aromatic groups prior to emission. 

J. R. Lakowicz and A. Baiter, Resolution of initially excited and relaxed states of tryptophan fluorescence by differential-wavelength deconvolution of time-resolved fluorescence decays, Biophys. Chem. 15, 353-360 (1982). 

The width of the 0-0 line in single-tryptophan proteins at 77 K has been interpreted to reflect inhomogeneous broadening arising because the protein exists as a distribution of conformations. 30 34 The width of the 0-0 band of liver alcohol dehydrogenase is 500 cm-1 at 22°C.(10 31 35) The widths of the 0-0 transition for other proteins are somewhat greater. In many cases for the spectra taken at room temperature, low-resolution optics were used (as in Figure 3.2), and hence the published spectra may overestimate the width of the emission band. 

If a collisional quencher of the fluorophore is also incorporated into the membrane, the lifetime will be shortened. The time resolution of the fluorescence anisotropy decay is then increased,(63) providing the collisional quenching itself does not alter the anisotropy decay. If the latter condition does not hold, this will be indicated by an inability to simultaneously fit the data measured at several different quencher concentrations to a single anisotropy decay process. This method has so far been applied to the case of tryptophans in proteins(63) but could potentially be extended to lipid-bound fluorophores in membranes. If the quencher distribution in the membrane differed from that of the fluorophore, it would also be possible to extract information on selected populations of fluorophores possibly locating in different membrane environments. 

Among protein aromatic groups, histidyl residues are the most metal reactive, followed by tryptophan, tyrosine, and phenylalanine.1 Copper is the most reactive metal, followed in order by nickel, cobalt, and zinc. These interactions are typically strongest in the pH range of 7.5 to 8.5, coincident with the titration of histidine. Because histidine is essentially uncharged at alkaline pH, complex-ation makes affected proteins more electropositive. Because of the alkaline optima for these interactions, their effects are most often observed on anion exchangers, where complexed forms tend to be retained more weakly than native protein. The effect may be substantial or it may be small, but even small differences may erode resolution enough to limit the usefulness of an assay. 

E.g. tryptophane residues of proteins excite at 290-295 mn but they emit photons somewhere between 310 and 350 mn. The missing energy is deposited in the tryptophane molecular enviromuent in the form of vibrational states. While the excitation process is complete in pico-seconds, the relaxation back to the initial state may take nano-seconds. While this period may appear very short, it is actually an extremely relevant time scale for proteins. Due to the inherent thermal energy, proteins move in their (aqueous) solution, they display both translational and rotational diffusion, and for both of these the characteristic time scale is nano-seconds for normal proteins. Thus we may excite the protein at time 0 and recollect some photons some nano seconds later. With the invention of lasers, as well as of very fast detectors, it is completely feasible to follow the protein relax back to its ground state with sub-nano second resolution. The relaxation process may be a simple exponential decay, although tryptophane of reasons we will not dwell on here display a multi-exponential decay. 

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... 

Besides having a noncovalent association of subunits as in tryptophan synthase, some enzymes are double-headed, in that they contain two distinct activities in a single polypeptide chain. A good example of this is the indole 3-glycerol phosphate-synthase-phosphoribosyl anthranilate isomerase bifunc-tional enzyme from the tryptophan operon of E. coli. The crystal structure of the complex has been solved at 2.0 A resolution.39 The two enzymes have been separated by genetic manipulation.40 The activity of the two separate monomeric monofunctional constituents is the same as in the covalent complex so there is no catalytic advantage of having the proteins fused. 

L-Tryptophan 10 Synthesis from acrylonitrile and resolution Second limiting amino acid for com... 

Since protein emission spectra are generally rather broad, larger emission bandwidths can usually be tolerated. Only where it is important to resolve tyrosine fluorescence from tryptophan fluorescence and the Rayleigh scattering peak is it necessary to minimize the bandwidth. In cases where only low concentrations of material are available, it is necessary to strike a balance between resolution and light intensity in order to obtain the best possible signal-to-noise ratio. Settings of 2.5 to 10 nm are normal. 

Varley, P.G., Dryden, D.T., and Pain, R.H. 1991. Resolution of the fluorescence of the buried tryptophan in yeast 3-phosphoglycerate kinase using succinimide. Biochim. Biophys. Acta 1077 19-24. 

The use of antibiotic-based CSPs has been reported in capillary electrochromatography (CEC) for chiral resolution [60]. Teicoplanin CSP covalently bonded to silica gel was used to resolve the enantiomers of tryptophan and dinitrobenzoyl leucine by CEC [61]. Good levels of enantioselectivity were obtained with optimized separations. Vancomycin covalently bonded to silica gel was also evaluated in CEC for the chiral resolution of thalidomide and jS-adrenergic blocking agents under all the three mobile phase modes. The... 

In 1973, Stewart and Doherty [9] resolved enantiomers of tryptophan on a column packed with BSA-succinoylaminoethyl-agarose in a discontinuous elution procedure. The mobile phase used was 0.1 M borate buffer (pH 9.2). The chromatograms of this classical research are shown in Figure 2. Several years later, this technique was applied for the chiral resolution of warfarin enantiomers [10]. In 1981, the enantiomers of tryptophan and warfarin racemates were resolved on various serum albumin CSPs [11,21,22]. The same method was used for the resolution of other drugs [12-14]. Allenmark et al. [23] studied the resolution of a series of active racemic sulfoxides on a BSA column using 0.08 M phosphate buffer (pH 5.8) as the eluting solvent. 

FIGURE 2 Enantiomeric resolution of DL-tryptophan on defated BSA-succinoyl-aminoethyl-agrose column (0.9x25cm) using borate buffer (0.1 M). (From Ref. 9.)... 

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