Amino acids spectrophotometric

For betaxanthins, partial synthesis is quite common and presents a viable tool for identification by co-injection experiments. - Starting from a red beet extract or semi-purified betanin-isobetanin blend, alkaline hydrolysis by addition of 32% ammonia is initiated. Spectrophotometric monitoring at 424 nm allows the release of betalamic acid to be followed. Betaxanthins are obtained through the addition of the respective amino acid or amine in at least 20-fold molar excess followed by careful evaporation. Since the starting material most often consists of a racemic betacyanin mixture, the resulting betaxanthin will also consist of two stereoisomers that may not easily be separated by HPLC. ... 

Application of the analytical techniques discussed thus far focuses upon detection of proteinaceous impurities. A variety of additional tests are undertaken that focus upon the active substance itself. These tests aim to confirm that the presumed active substance observed by electrophoresis, HPLC, etc. is indeed the active substance, and that its primary sequence (and, to a lesser extent, higher orders of structure) conform to licensed product specification. Tests performed to verify the product identity include amino acid analysis, peptide mapping, N-terminal sequencing and spectrophotometric analyses. 

An extractive spectrophotometric procedure based on the complexation of reduced Iron(II) with 5-Chloro-7-iodo-8-hydroxyquinoline (CIHQ) for the estimation of micro amounts of vitamin C. The resulting brown colored complex was extracted into chloroform to give a reddish brown extract which shows an absorption band at 485 nm. This chelate was formed immediately and the apparent molar absorptivity and Sandell s sensitivity for vitamin C was found to be 8.5 x 105 dm3 mol"1 cm 1 and 2.072xl0 4g cm 2. Linear relationship between absorbance and concentration of ascorbic acid is observed up to 0.8 pg ml"1. Interference studies of different substances including sugars, vitamins and amino acids, metal ions and organic acids were carried out. The utility of the method was tested by analysing some of the marketed products of vitamin C... 

Depending on the derivatizing agent used, spectrophotometric or fluorometric detectors are usually employed. Electrochemical detection of underivatized amino acids is limited to amino acids possessing aromatic or sulfur-containing side-chain, even if derivatization procedmes to attach electrochemical active moieties to the amino acids can be employed, as well as other approaches... 

As for Boc amino acids, all proteinogenic and several non-natural Fmoc amino acids esterified with Wang or similar resins are commercially available. Deprotection with 20% piperidine in DMF is usually complete within a few minutes, and the release of the fluorene derivative is easily monitored spectrophotometrically at 300-320 nm (see, e.g., [33]). The peak area can be used to determine the amount of chromophore released, which is proportional to the efficiency of the preceding coupling reaction. At the end of an automated peptide synthesis, inspection of the chromatogram of all Fmoc releases enables rapid assessment of the quality of the resin-bound peptide and quick location of positions in the peptide where coupling was unsuccessful or difficult. 

Biochemical research often requires the quantitative measurement of protein concentrations in solutions. Several techniques have been developed however, most have limitations because either they are not sensitive enough or they are based on reactions with specific amino acids in the protein. Since the amino acid content varies from protein to protein, no single assay will be suitable for all proteins. In this section we discuss five assays three older, classical methods that are occasionally used today and two newer methods that are widely used. In four of the methods, chemical reagents are added to protein solutions to develop a color whose intensity is measured in a spectrophotometer. A standard protein of known concentration is also treated with the same reagents and a calibration curve is constructed. The other assay relies on a direct spectrophotometric measurement. None of the methods is perfect because each is dependent on the amino acid content of the protein. However, each will provide a satisfactory result if the proper experimental conditions are used and/or a suitable standard protein is chosen. Other important factors in method selection include the sensitivity and accuracy desired, the presence of interfering substances, and the time available for the assay. The various methods are compared in Table 2.3. 

B 9. What amino acid residues are detected when the spectrophotometric assay is used to quantify proteins Are those amino acids present in the same quantity in all proteins Explain how this may affect measurement of proteins by this method. 

Ideally, for purified or partially purified protein, the protein standard should have an aromatic amino acid content similar to that of the sample protein. For the total protein of a crude lysate, bovine serum albumin (BSA) is a commonly used standard for spectrophotometric quantitation of protein concentration. A 3 mg/ml solution of BSA should have an A280 of 1.98, based on an A2S0 of 6.61 for a 1% (w/v) solution. 

Picric acid Suitable for extraction of amino acids and small peptides. Interferes with Kjeldahl or spectrophotometric methods Fox (1989), Salji and Kroger (1981), and Reville and Fox (1978)... 

Many useful compounds such as amino acids, nucleic acids, alcohols, vitamins, antibiotics, foods, etc. are produced in fermentation industries. Furthermore, many organic and inorganic compounds are present in waste waters. The determination of these compounds is required for control of fermentation and environment. Analysis of these compounds can be done by spectrophotometric methods. However, complicated procedures and long reaction times are required. 

Additionally the concentration of the amino acid has been determined by a spectrophotometric method using... 

Mixed N- and O-Donor Ligands. Spectrophotometric titration methods have been used to establish the existence and stoicheiometry of V111 complexes with alanine, a-aminobutyric acid, lysine, and glycylglycine pH measurements showed that the amino-acid ligand is co-ordinated in the anion form.385... 

Pathway C seemed to be especially attractive, because it should enable addition of acyl anion equivalents to a large number of readily accessible activated carboxylic acids (Figure 3.6.10). Thus diversity in all relevant positions should be readily attainable. High-loaded triphenyl phosphine resin 12 (1.6 mmol g-1) was alkylated with bromoacetonitrile under the action of microwave irradiation yielding phos-phonium salt 13 quantitatively. 13 was converted into stable ylide 14 by treatment with tertiary amine. Carboxylic acids were activated in the presence of N-(3-dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride (EDC) and reacted with 14 yielding acyl cyanophosphoranes 15. The reaction was monitored by ATR-IR coupling yields could be determined by spectrophotometric Fmoc-determination and were 90% for Fmoc-phenylalanine as reference amino acid. 

The chromophoric pyridoxal phosphate coenzyme provides a useful spectrophotometric probe of catalytic events and of conformational changes that occur at the pyridoxal phosphate site of the P subunit and of the aiPi complex. Tryptophan synthase belongs to a class of pyridoxal phosphate enzymes that catalyze /3-replacement and / -elimination reactions.3 The reactions proceed through a series of pyridoxal phosphate-substrate intermediates (Fig. 7.6) that have characteristic spectral properties. Steady-state and rapid kinetic studies of the P subunit and of the aiPi complex in solution have demonstrated the formation and disappearance of these intermediates.73-90 Fig. 7.7 illustrates the use of rapid-scanning stopped-flow UV-visible spectroscopy to investigate the effects of single amino acid substitutions in the a subunit on the rate of reactions of L-serine at the active site of the P subunit.89 Formation of enzyme-substrate intermediates has also been observed with the 012P2 complex in the crystalline state.91 ... 

A small peptide was subjected to hydrolysis and amino acid analysis. In addition, because acid hydrolysis destroys tryptophan, the tryptophan content was estimated spectrophotometrically. From the following data, determine the empirical formula of the peptide. 

Ultraviolet spectrophotometric analysis of the incorporation or deblocking of the a,a-dimethyl-3,5-dimethoxybenzyloxycarbonyl (Ddz) group in solid phase synthesis has been used by Birr for the analysis of the coupling and deprotection reactions respectively 18 20). Similar spectroscopic properties of the N-protecting groups have been used for the analysis in the solid phase synthesis involving Nps-amino acids 21,22) and Bpoe-amino acids 23). In all these methods the uptake or release of a soluble reagent or byproduct is analyzed. 

As the amino acids come off the column they are accessed by the ninhydrin reagent. The ninhydrin-amino acid complexes (Schemes 7.3 and 7.4) are detected spectrophotometrically at 570 nm (primary amino acids) and 440 nm (secondary amino acids, such as proline) and recorded as a series of peaks (Fig. 7.6). The retention time of the peak on the chart identifies the amino acid, the area under the peak indicating the quantity of the amino acid present. 

Within the limits of error of amino acid analyses available at the time, the count of groups obtained by Cannan et al. agreed with expectation, except in so far as the alkaline part of the curve was concerned. The number of groups titrated here is essentially the same as the number of amino groups, rather than the sum of amino and phenolic groups. This result is in accord with the later spectrophotometric titration of phenohc groups essentially all of these groups are inaccessible to titration in the native protein. 

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