Quantitative amino-acid analysis

HPLC and GLC of derivatised amino acids are overtaking the classical amino acid analyser (the Moore and Stein ion-exchange separation plus post-column ninhy- 

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The amino add analysis of all peptide chains on the resins indicated a ratio of Pro Val 6.6 6.0 (calcd. 6 6). The peptides were then cleaved from the resin with 30% HBr in acetic acid and chromatogra phed on sephadex LH-20 in 0.001 M HCl. 335 mg dodecapeptide was isolated. Hydrolysis followed by quantitative amino acid analysis gave a ratio of Pro Val - 6.0 5.6 (calcd. 6 6). Cycll2ation in DMF with Woodward s reagent K (see scheme below) yielded after purification 138 mg of needles of the desired cyc-lododecapeptide with one equiv of acetic add. The compound yielded a yellow adduct with potassium picrate, and here an analytically more acceptable ratio Pro Val of 1.03 1.00 (calcd. 1 1) was found. The mass spectrum contained a molecular ion peak. No other spectral measurements (lack of ORD, NMR) have been reported. For a thirty-six step synthesis in which each step may cause side-reaaions the characterization of the final product should, of course, be more elaborate. 

Larsen, B. R. and West, F. G., A method for quantitative amino acid analysis using precolumn o-phthaladehyde derivatization and high performance liquid chromatography, /. Chromatogr. Sci., 19, 259, 1981. 

S. Keck, T. Peters, Identification of protein containing paint media by quantitative amino acid analysis, Studies in Conservation, 14, 75 82 (1969). 

The Number of Tryptophan Residues in Bovine Serum Albumin A quantitative amino acid analysis reveals that bovine serum albumin (BSA) contains 0.58% tryptophan (Afr 204) by weight. 

RG Elkin. Quantitative amino acid analysis of feedstuff hydrolysates by reverse phase liquid chromatography and conventional ion-exchange chromatography. J Assoc Off Anal Chem 67 1024-... 

The subunit stoichiometry for the coated vesicle enzyme has been determined by quantitative amino acid analysis (Arai et al, 1988 Xu et al, 1999). 

A stock solution of rabbit Cytochrome c was made up in water at a concentration of 4.3 mg/ml. The concentration of the stock solution was determined by quantitative amino acid analysis of a 5pJ aliquot that was hydrolyzed and analyzed as previously described (4). The stock solution was diluted 5-fold with 0. IM ammonium bicarbonate or 0. IM sodium borate (both at pH 8.2) and TPCK-Trypsin in lOmM HCl was added to give a 1 20 enzyme protein solution (w/w). Digestion was carried out at 37°C for 24 hours after which the enzymatic activity was terminated by heating at 100°C for 5 min. 

The overall amino acid composition is determined using various hydrolytic and analytical procedures and compared with the amino acid composition deduced from the gene sequence for the desired product, or the natural counterpart, if considered necessary. In many cases, amino acid composition analysis provides some useful structural information for peptides and small proteins, but such data are generally less definitive for large proteins. Quantitative amino acid analysis data can also be used to determine protein content in many cases. 

The degree of glycosylation of the glycopolypeptides was estimated via H NMR, MALDI-TOF, and periodate assay (Glycoprotein Carbohydrate Estimation Kit (Pierce, Rockford, IL). H NMR spectra were acquired on a Bruker DRX-400 NMR spectrometer under standard quantitative conditions at 25 °C, and the standard protocols for the periodate assay were as described by the manufacturer. Comparison of sample solutions to a calibration curve of proteins of known carbohydrate content, combined with quantitative amino acid analysis of the samples, permitted estimation of the degree of substitution of the glycopolypeptides. 

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. 

The molecular weight of tyrocidine A was found to be about 1,270 . Total hydrolysis and quantitative amino acid analysis revealed the exact composition . Partial hydrolysis, fractionation of the resulting peptide mixture by countercurrent distribution, ion-exchange chromatography and paper-chromatography, followed by sequential analysis, led Paladini and Craig to the structure of tyrocidine A see Table 1.6). 

The molecular weight of polymyxin B was shown by the method of partial substitution to be 1,150 d 10 per cent. Quantitative amino acid analysis yielded the amino acids a,y-diaminobutyric acid, L-threonine, D-phenylalanine and L-leucine in the molar proportions 6 2 1 1. Because no free a-carboxyl- and no free a-amino groups could be detected, polymyxin Bi had to be of a cyclic nature . Neither pepsin nor trypsin was found to attack the molecule and therefore partial hydrolysis and separation of the fragments was used for the elucidation of the structure. Of the 14 fragments isolated and identified, seven key peptides were necessary for proposing four tentative structures for polymyxin Two were formulated with a ring... 

Molecular weight determination by the method of partial substitutional and spectrophotometric measurements of picrates gave values of about 1,360 for the hydrochlorides of both colistin A and B . Quantitative amino acid analysis showed the presence of six moles of L-a,y-diaminobutyric acid, two moles of L-threonine, one mole of L-leucine and one mole of D-leucine in both colistin A and By partial acid hydrolysis as well as by enzymatic... 

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). 

Quantitative amino acid analysis can be used to determine the loading of a resin. Following esterification of the first amino acid, the amino acid-resin is hydrolyzed with a 12 N HCl-propionic acid (1 1) solution for 90 min at... 

Many physicochemical assays are established to quantify the protein mass. It is determined by exploiting the extinction coefficient in optical density measurements or by colorimetric assays such as the Bradford, Lowry, bicinchoninic (BCA), and biuret assay [13, 14]. Albeit easy to perform, these colorimetric assays suffer from inaccuracies that are due to the use of inappropriate standards like bovine serum albumin. If relevant standards are not available, quantitative amino acid analysis [6], the (micro-)Kjeldahl nitrogen method [14, 15] or gravimetry as very accurate but time-consuming alternatives can be applied. 

Amino acid analysis, determination of amino acid composition of a peptide by complete hydrolysis followed by the quantitative analysis of the liberated amino acids. For hydrolysis, numerous chemical and enzymatic protocols are known. Based on the pioneering studies of Stein and Moore, amino acid analysis has been automated. Nowadays, instmments are in use for quantitative amino acid analysis which are based on partition chromatography, such as HPLC and gas-liquid chromatography [S. Blackburn, Amino Add Determination, M. Dekker, New York, 1978 ... 

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