Leucine separation from isoleucine

NAD+ serves as the oxidant. The reaction is catalyzed by a complex of enzymes whose molecular mass varies from 4 to 10 x 106, depending on the species and exact substrate.297 Separate oxoacid dehydrogenase systems are known for pyruvate,298-300 2-oxoglut-arate,301 and the 2-oxoacids with branched side chains derived metabolically from leucine, isoleucine, and... 

Figure 4 HPLC separation of 17 PTC AAs (Pico-Tag column 150 x 3.9mm, eluent A 0.14moll NaAc, pH 6.4, containing 0.5ml TEAI B 60% acetonitrile in water flow rate 1 ml minpeaks 1 = aspartic, 2=glutamic acids, 3 = serine, 4=glycine, 5=his-istidine, 6 = arginine, 7=threonine, 8 = alanine, 9 = proline, 10 = ammonia, l1=tyrosine, 12=valine, 13=methionine, 14=cystine, 15 = isoleucine, 16 = r>leucine, 17 = phenylalanine, 18=tryptophan. (Reproduced with permission from Bidlingmayer BA ef a/. (1984) Journal of Chromatography 336 Elsevier.)...

Figure 5 Separation of 27 PTC AAs (column 150+ (20 guard) x 4 mm, Cl 8 Hypersil Sum, T=50°C, eluent A 0.05 mol I" NaAc, pH 7.2, B A eluent/acetonitrile/methanol = 46/44/10 (pH 7.2) flow rate 2.1 ml min peaks 1 = aspartic, 2 = glutamic acids, 3 = hy-hydroxyproline, 4 = serine, 5 = glycine, 6 = asparagine, 7 = -alanine, 8 = glutamine, 9 = homoserine, 10 = y-aminobutyric acid (GABA), 11 =histidine, 12 = threonine, 13 = alanine, 14 = 1-amino-1-cyclopropane carboxylic acid (ACPCA), 15 = arginine, 16 = proline, 17 = homoarginine, 18 = tyrosine, 19 = valine, 20 = methionine, 21 =cyst(e)ine, 22 = isoleucine, 23=n-leucine, 24 = phenyl-ylalanine, 25 = tryptophan, 26 = ornithine, 27 = lysine = system peaks. (Reproduced with permission from Vasanits A and Molnar-Perl I (2000) Journal of Chromatography A 870 271-287 Elsevier.)...

Rudman and Meister IJ ) first showed the presence of a transaminase in cell-free extracts of E. colt that catalyze transamination reactions between glutamate and isoleucine, valine, leucine, norleucine, and norvaline. These monocarboxylic amino acids transaminated with each other as well as with glutamine. Preparations of an E. cdi mutant which did not respond to a-keto- 8-methylvalerate was unable to transaminate isoleucine or valine. The transaminase responsible for activity with the branched-chain amino acids was separated from other transaminases and considerably purified by standard methods of protein purification. It was shown to... 

Lipoproteins are proteins associated with transport of lipids. As an example, apolipoprotein BlOO (apo BlOO) is synthesized in the liver and used to transport very low density lipoprotein (VLDL) particles containing triglycerides and cholesterol from the hepatocyte into the systemic circulation. The rate of VLDL secretion is quantifled by labeling the apo BlOO moiety via intravenous infusion of labeled amino acid, such as leucine or valine, and measurement of C enrichment in the leucine or valine present in the VLDL apo BlOO. GG-MS and GC C IRMS can be applied [40,86-90]. Because of the difficult separation of leucine and isoleucine, valine is chosen as the preferred substrate in conjunction with GC-C-IRMS [91,92]. 

Leucine.—The greater part of the leucine is contained in the ester fractions, which boil between 70° and 90° C. It generally occurs in considerable amounts in the protein, and is obtained by crystallisation from water, in which it is less soluble than the other amino acids which may be present. It is seldom present in its pure, optically active form, as this is easily racemised, and the various crops of crystals most probably also contain isoleucine. It is more easily isolated by completely racemis-ing the mixture of amino acids contained in this fraction by heating in an autoclave with baryta to 160-180° C., and then, after removal of the baryta, separating it by crystallisation. The difficulty of separating it from the other amino acids, especially valine and isoleucine, makes an exact quantitative estimation almost impossible. The values which have been found are therefore minimal ones, and they will also include in many cases the yield of isoleucine. 

Fig.4.40. Separation of some DNS-amino acids on a small-particle silica-gel column (see text for details). Peaks 1 = inert 2 = unknown 3 isoleucine 4 = valine 5 = leucine 6 = tyrosine 7 = alanine 8 = tryptophan 9 = glycine 10 = histidine 11= lysine. (From ref. 81 with permission of the American Chemical Society, Washington.)...

Fig. 8.15. Effect of particle diameter and pore diameter on the separation of 12 PTH-amino acids. Column, 150 x 0.075 mm i.d. packed with 6 pm/300 A or (b) 3.5 pm/80 A Zorbax ODS eluents, (A) 2 mmol/1 ammonium acetate, pH 7.0, (B) 2 mmol/1 ammonium acetate, pH 7.0, 90% acetonitrile gradient elution with 30- 90% B in 15 min flow rate of mobile phase through inlet reservoir, 100 pl/min applied voltage, 20 kV detection, ESI-MS, 0.5 s/spectrum integration time sheath liquid, 1 mmol/1 ammonium acetate, pH 7.0, 90% methanol, 3 pl/min injection, electrokinetic, 2 kV, 2 s sample, PTH-asparagine, PTH-glutamine, PTH-threonine, PTH-glycine, PTH-alanine PTH-tyrosine, PTH-valine, PTH-proline PTH-tryptophan, PTH-phenylalanine, PTH-isoleucine, PTH-leucine (in order of elution). (Reproduced from ref. [113] with permission of the author).

The exact location of the Schiff base and the Asp-96 carboxyl is yet to be found since the three-dimensional structure of bacteriorhodopsin is not known to atomic resolution. Electron microscopy of two-dimensional bacteriorhodopsin crystals indicates that the Schiff base is localized in the middle of the protein molecule, while Asp-96 is somewhere between the Schiff base and the cytoplasmic surface of the membrane [21]. It has also been shown that the protein regions separating the Schiff base from the outer and cytoplasmic membrane surfaces differ strongly in hydrophobicity, which is low in the former, and high in the latter case. Between the outer membrane surface and the Schiff base, there are four charged amino acids and no valine, leucine and isoleucine. At the same time, between the Schiff base and the cytoplasm, five leucines, valine and only one charged amino acid (Asp-96) seem to be localized [21]. Thus the dielectric... 

Proton resonance spectra of denatured proteins consist of sharp peaks which correspond to a summation of resonances from individual residues [129]. In C spectra of denatured proteins, it is possible to distinguish all the carbon resonances of the aromatic side chains of histidine, phenylalanine, tyrosine and tryptophan, and separate resonances from alanine, arginine, glycine, isoleucine, leucine, threonine, valine and occasionally methionine [130]. (A natural abundance C spectrum of a 13 mM solution of lysozyme takes only 4 hr accumulation time using 20 mm sample tubes [131]). 

These data were reintroduced to the ANN as another verihcation point. The response surface was similar to that generated from experiment 11. This indicated that the network had sufficient data and had reached its operation limits. Therefore, optimum 2 provided the best separation that was possible using this separation electrolyte. It should be noted isoleucine and leucine, and histidine and proline comigrated under all experimental conditions that were investigated. Optimum 3 was also investigated and as the response surface indicated, the resolution was not as good as optimum 2 (Fig. 7.8). 

The circulins—As early as 1949, Peterson and Reineke characterized circulin as its sulphate. Total hydrolysis yielded D-leucine, L-threonine and L-K,y-diaminobutyric acid together with an optically active isomer of pelargonic acid. The existence of two components, found by Peterson and Reineke was later confirmed by the chromatographic separation of crude circulin into two major components, named circulin A and circulin B. In addition there was evidence for at least three other ninhydrin-positive, biologically active entities. In the hydrolysate of circulin A, L-isoleucine was found besides the amino acids previously reported . Quantitative amino acid analysis showed circulin A and B to be composed of L-a,y-diamino-butyric acid, L-threonine, D-leucine, L-isoleucine and ( + )-6-methyloctanoic acid in the molar ratio 6 2 1 1 1. After partial acid hydrolysis, fractionation and structure determination of the resulting peptides, circulin A and circulin B were formulated as cyclodecapeptides . Very recently, however, Japanese workers have revised the structure of circulin A. According to them, circulin A differs from colistin A only by a replacement of L-leucine in the latter by L-isoleucine Figure 1.7). 

Fig. 4. Chromatogram of separation on a silicone stationary phase of methyl esters of trifluoro-acetylated amino acids of hydrolysate of human fingernail. Sorbent silicone stationary phase. Temperature programme A, 100°C, isothermal B, heating from 100°C at 1.5°C/min C, heating from 116.5°C at 4°C/min D, 140°C, isothermal E, heating from 140°C at 6°C/min to 210°C. Peaks 1 = alanine 2 = valine 3 = glycine 4 = isoleucine 5 = threonine 6 = leucine 7 = norleucine 8 = internal standard 9 = proline 10 = asparagine 11 = glutamine 12 = phenylalanine 13 = tyrosine 14 = lysine. From ref. 13.

Fig. 4.4.19. Separation of amino acids on a cation-exchange resin and with post-column derivation with ninhydrin. B was obtained 50 analyses after A. Peak identification D, aspartic acid T. threonine S, serine E. glutamic acid P. proiine G, glycine A. alanine C. cysteine V, valine M. methionine I, isoleucine L. leucine NL. norleucine F. phenylalanine O. ornithine K, lysine NH,. ammonia H. histidine R. arginine W. tryptophan. Reprinted from Ref. 96 with permission.

Fig. 11.2.11. Isocratic separation of PTH-amino adds. Chromatographic conditions column, Ultrasphere ODS (250 X 4.6 mm I.D.) mobile phase, 0.01 M sodium acetate (pH 4.9)-acetonitrile (62.2 37.8) flow rate, 1 ml/min temperature, ambient. Peak identity corresponding to the single letter code for amino acids D, aspartic acid E, glutamic acid N, asparagine Q, glutamine T, threonine G, glycine A, alanine Y, tyrosine M, methionine V, valine P, proline W, tryptophan F, phenylalanine K, lysine I, isoleucine L, leucine S, serine. Reproduced from Noyes (1983), with...

Fig. 11.2.12. Normal phase separation of amino acids. Chromatographic conditions column, Zorbax NH2 (250 x 4.6 mm I.D.) mobile phase, 10 mM potassium phosphate, pH 4.3 (A), acetonitrile-water 50 7 (v/v) (B) flow rate, 2 ml/min temperature, 35 °C. Peaks 1, phenylalanine 2, leucine 3, isoleucine 4, methionine 5, tyrosine 6, valine 7, proline 8, alanine 9, hypro 10, threonine 11, glycine 12, serine 13, histidine 14, cysteine 15, arginine 16, lysine 17, hydroxylysine 18, glutamic acid 19, aspartic acid. Reproduced from Smolensk et al. (1983), with permission.

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