Amino enzymatic analysis

ENZYMATIC ANALYSIS WITH CARBOXYPEPTIDASES. Carboxypeptidases are enzymes that cleave amino acid residues from the C-termini of polypeptides in a successive fashion. Four carboxypeptidases are in general use A, B, C, and Y. Carboxypeptidase A (from bovine pancreas) works well in hydrolyzing the C-terminal peptide bond of all residues except proline, arginine, and lysine. The analogous enzyme from hog pancreas, carboxypeptidase B, is effective only when Arg or Lys are the C-terminal residues. Thus, a mixture of carboxypeptidases A and B liberates any C-terminal amino acid except proline. Carboxypeptidase C from citrus leaves and carboxypeptidase Y from yeast act on any C-terminal residue. Because the nature of the amino acid residue at the end often determines the rate at which it is cleaved and because these enzymes remove residues successively, care must be taken in interpreting results. Carboxypeptidase Y cleavage has been adapted to an automated protocol analogous to that used in Edman sequenators. 

As the name implies, the odor of urine in maple syrup urine disease (brancbed-chain ketonuria) suggests maple symp or burnt sugar. The biochemical defect involves the a-keto acid decarboxylase complex (reaction 2, Figure 30-19). Plasma and urinary levels of leucine, isoleucine, valine, a-keto acids, and a-hydroxy acids (reduced a-keto acids) are elevated. The mechanism of toxicity is unknown. Early diagnosis, especially prior to 1 week of age, employs enzymatic analysis. Prompt replacement of dietary protein by an amino acid mixture that lacks leucine, isoleucine, and valine averts brain damage and early mortality. 

A3. Albanese, A. A., Davis, V. I., Smetak, E. M., Lein, M., and Fisher, M., Amino acid analysis of the nondiffusible fraction of enzymatic protein digests and human urine. Arch. Biochem. Biophys. 20, 47-54 (1949). 

The reduced form of Na+, K+-ATPase inhibitor-I (10) was obtained by treatment of the protected peptide synthesized by the soln procedure with HF, followed by reaction with Hg(OAc)2. After purification of the crude product on Sephadex G-25, the reduced peptide (110 mg) was dissolved in 0.1 M NH4OAc buffer (1L, pH 7.8) at a peptide concentration of 0.018 mM and then stirred at rt. After 24 h, the major peak in the HPLC, which coeluted with the natural product, corresponded to 55% of the product distribution. The mixture was acidified to pH 3 with AcOH and 10 was purified by RP-HPLC. When the oxidation was carried out in the presence redox reagents at a peptide/GSH/GSSG ratio of 1 100 10, after 24 h the major oxidation product increased to 69%. The mixture was acidified with AcOH and the product (10) isolated by preparative HPLC yield 20%. The product was characterized by MALDI-TOF-MS and amino acid analysis a combination of enzymatic peptide mapping and synthetic approaches were applied to assign the cystine connectivities. 

For the analytical characterization of sulfated tyrosine peptides, spectroscopic methods as well as amino acid analysis and, more recently, mass spectrometry are employed. In Table 2 the spectroscopic data of tyrosine 0-sulfate are compared to those of the related sulfonic acid derivatives as possible byproducts in the chemical sulfation of the tyrosine or tyrosine peptides.[361 In the course of the synthesis of tyrosine 0-sulfate peptides and, particularly in the final deprotection step, desulfation may occur which limits the characterization of the final compounds in terms of quantitative identification of the tyrosine 0-sulfate. This is achieved by amino acid analyses of basic hydrolysates of the sulfated tyrosine peptides or preferably by analyses of the enzymatic hydrolysates with aminopeptidase M or leucine-aminopeptidase. 

Volkmann S, Wohrl BM, Tisdale M, Moelling K. Enzymatic analysis of two HIV-1 reverse transcriptase mutants with mutations in carboxyl-terminal amino acid residues conserved among retroviral ribonucleoside H. JBiol Chem 1993 268 2674-2683. 

M Offenzeller, G Santer, K Totschnig, Z Su, H Moser, R Traber, E Schneider-Scherzer. Biosynthesis of the unusual amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine of cyclosporin A enzymatic analysis of the reaction sequence including identification of the methylation precursor in a polyketide pathway. Biochemistry 35 8401-8412, 1996. 

The HPLC (High Performance Liquid Chromatography) analysis of enzymatic bath was performed using a chromatographic column Cl8 DABS (Beckman, USA) modified to amino acid analysis. 

Jones, B. N., Paabo, S., and Stein, S. (1981). Amino acid analysis and enzymatic sequence determination of peptides by an improved o-phthaldialdehyde precolumn labeling procedure. J. Liq. Chro-matogr. 4, 565-586. 

IPG and RP-HPLC were useful for separating several monoacetylated forms of rpST but these methods do not yield quantitation for the total level of e-N-acetyllysine. The total amount of e-N-acetyllysine was determined using total enzymatic digestion of the protein followed by amino acid analysis. Normal hydrolysis with acid or base cannot be used since e-N-acetyllysine is not stable to these hydrolysis conditions. A value of 0.42 moles of e-N-acetyllysine per mole of protein was obtained for the low pl rpST (Fig. 2, lane 4) while the normal pi rpST (Fig. 2, lane 3) yielded non-detectable levels (<0.05 mol/mol) of this amino acid. As expected, these data confirmed that e-N-acetyllysine is present only in the low pi components of rpST but not in the normal pi rpST. 

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 hydrolysis of proteins by chemical and enzymatic methods is discussed by R. L. Hill in the second chapter. The chapter should serve as a standard source for information on the many operational techniques with which only the experienced specialist is likely to be familiar. Dr. Hill covers not only the partial hydrolysis of proteins by general and specific procedures but also tbe total enzymatic hydrolysis of proteins for the purpose of amino acid analysis. 

Pyr-His-Trp-OEt (21 g, 43.7 mmol) and H-Ser-Tyr-o-Phe-Leu-Arg-Pro-Gly-NHj (20 g, 23.9 mmol) were suspended in a mixture of DMF (66 mL) and H2O (70.5 mL) the pH being adjusted to 8.0 by addition of 2M KOH. The reaction was started by addition of a-ch5miotr5 psin (50 mg). After 6.5 h, AcOH (10 mL) was added to stop the enzymatic reaction. After concentration, precipitation by addition of acetone gave [D-Phe ]LHRH yield 24.4 g. The crude peptide was purified by RP chromatography to give the LHRH peptide which was characterized by HPLC (fR = 15.5min, 99%) and amino acid analysis 5deld 16.2g (53%). 

The sequence of amino acids in the polypeptide chain (i.e., the primary structure of a polypeptide or protein) can be established by selective chemical and enzymatic cleavage of the protein followed by separation, amino acid analysis, and sequence determination of all peptide fragments. The entire amino acid sequence is established by overlapping identical regions of the individual fragments. The following problem illustrates the procedure. 

Amino acid analysis is widely applied in research, clinical facilities, and industry. It is a fundamental technique in biotechnology, used to determine the concentration of peptide solutions, to confirm protein binding in antibody conjugates, and for end-terminal analysis following enzymatic digestion. Clinical applications include diagnosing metabolic disorders in newborns. In industry, it is used for quality control of products ranging from animal feed to infant formula. 

Enzymatic Methods in Amino Acid Analysis. Annals New York Acad. 

Modification of the prcformulation format for biotechnological products from the original guidance must be considered. The sections regarding chemical structure, physicochemical properties, and stability may be revised according to the nature and characteristics of proteins and peptides. Aside from the conventional analytical instruments and techniques used in the study of small molecules, methods such as amino acid analysis, sequence analysis bioassay, immunoassay, and enzymatic assay are commonly used and should be included in the report. 

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... 

No other cofactors are required for enzymatic catalysts of this reaction. Amino acid analysis on isolated isoenzymes I and III indicate that the lower molecular weight monomers from I are not proteolytic degradation products of the monomers from III [68], When dimers of I (a ) are mixed with dimers of III ()8y8) an equilibrium concentration of II (afi) is formed. 

Oligonucleotide-peptide conjugates are characterized by UV spectrophotometry, amino acid analysis [71], nucleoside analysis after enzymatic degradation and mass spectrometry [72,73]. 

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 ... 

Peptide hydrolysis, complete hydrolysis of peptides and proteins for amino acid analysis or the production of individual amino acids from the peptide or protein hydrolysate. For this purpose, numerous chemical and enzymatic protocols are known, but none of these procedures alone is fully satisfactory. Besides hydrolysis with 6 M hydrochloric acid at 120 °C for 12 h, or with dilute alkali (2-4 M NaOH) at 100 °C for 4-8 h, mixtures of peptidases can also be used for complete peptide hydrolysis. Restricted or limited peptide hydrolysis ( peptide cleavage) is important for - sequence analysis and peptide mapping. 

A well-established conventional protocol to locate disulfide bonds in proteins includes cleaving a protein between the half-cysteinyl residues in a manner that leaves the disulfide bonds intact, isolating the cysteine-containing peptides, and performing either amino acid analysis or sequence analysis [2]. Alternatively, the disulfide bonds are reduced sequentially to thiols and alkylated with radiolabeled iodoacetic acid [1,3]. The completely reduced protein is cleaved enzymatically, and the disulfide bonds are detected by identifying the radiolabeled peptides. 

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