GLOBAL DOCUMENT
AMINO ACID ANALYSIS
Amino acid analysis refers to the methodology used to determine the amino acid composition or content of proteins, peptides, and other pharmaceutical preparations. Proteins and peptides are macromolecules consisting of covalently bonded amino acid residues organized as a linear polymer. The sequence of the amino acids in a protein or peptide determines the properties of the molecule. Proteins are considered large molecules that commonly exist as folded structures with a specific conformation, while peptides are smaller and may consist of only a few amino acids. Amino acid analysis can be used to quantify protein and peptides, to determine the identity of proteins or peptides based on their amino acid composition, to support protein and peptide structure analysis, to evaluate fragmentation strategies for peptide mapping, and to detect atypical amino acids that might be present in a protein or peptide. It is necessary to hydrolyze a protein/peptide to its individual amino acid constituents before amino acid analysis. Following protein/peptide hydrolysis, the amino acid analysis procedure can be the same as that practiced for free amino acids in other pharmaceutical preparations. The amino acid constituents of the test sample are typically derivatized for analysis.
Apparatus
Methods used for amino acid analysis are usually based on a chromatographic separation of the amino acids present in the test sample. Current techniques take advantage of the automated chromatographic instrumentation designed for analytical methodologies. An amino acid analysis instrument will typically be a low-pressure or high-pressure liquid chromatograph capable of generating mobile phase gradients that separate the amino acid analytes on a chromatographic column. The instrument must have postcolumn derivatization2
capability, unless the sample is analyzed using precolumn derivatization. The detector is usually an ultraviolet-visible or fluorescence detector depending on the derivatization method used. A recording device (e.g., integrator) is used for transforming the analog signal from the detector and for quantitation. It is preferred that instrumentation be dedicated particularly for amino acid analysis.
General Precautions
Background contamination is always a concern for the analyst in performing amino acid analysis. High purity reagents are necessary (e.g., low purity hydrochloric acid can contribute to glycine contamination). Analytical reagents are changed routinely every few weeks using only high-pressure liquid chromatography (HPLC) grade solvents. Potential microbial contamination and foreign material that might be present in the solvents are reduced by filtering solvents before use, keeping solvent reservoirs covered, and not placing amino acid analysis instrumentation in direct sunlight.
Laboratory practices can determine the quality of the amino acid analysis. Place the instrumentation in a low traffic area of the laboratory. Keep the laboratory clean. Clean and calibrate pipets according to a maintenance schedule. Keep pipet tips in a covered box; the analysts may not handle pipet tips with their hands. The analysts may wear powder-free latex or equivalent gloves. Limit the number of times a test sample vial is opened and closed because dust can contribute to elevated levels of glycine, serine, and alanine.
A well-maintained instrument is necessary for acceptable amino acid analysis results. If the instrument is used on a routine basis, it is to be checked daily for leaks, detector and lamp stability, and the ability of the column to maintain resolution of the individual amino acids. Clean or replace all instrument filters and other maintenance items on a routine schedule.
Reference Standard Material
Acceptable amino acid standards are commercially available for amino acid analysis and typically consist of an aqueous mixture of amino acids. When 3
determining amino acid composition, protein or peptide standards are analyzed with the test material as a control to demonstrate the integrity of the entire procedure. Highly purified bovine serum albumin has been used as a protein standard for this purpose.
Calibration of Instrumentation
Calibration of amino acid analysis instrumentation typically involves analyzing the amino acid standard, which consists of a mixture of amino acids at a number of concentrations, to determine the response factor and range of analysis for each amino acid. The concentration of each amino acid in the standard is known. In the calibration procedure, the analyst dilutes the amino acid standard to several different analyte levels within the expected linear range .................More Read....
EC 6. Ligases
EC 6.1 Forming Carbon-Oxygen Bonds
EC 6.1.1 Ligases Forming Aminoacyl-tRNA and Related Compounds
EC 6.1.1.1 tyrosine—tRNA ligase
EC 6.1.1.2 tryptophan—tRNA ligase
EC 6.1.1.3 threonine—tRNA ligase
EC 6.1.1.4 leucine—tRNA ligase
EC 6.1.1.5 isoleucine—tRNA ligase
EC 6.1.1.6 lysine—tRNA ligase
EC 6.1.1.7 alanine—tRNA ligase
EC 6.1.1.8 deleted
EC 6.1.1.9 valine—tRNA ligase
EC 6.1.1.10 methionine—tRNA ligase
EC 6.1.1.11 serine—tRNA ligase
EC 6.1.1.12 aspartate—tRNA ligase
EC 6.1.1.13 D-alanine-poly(phosphoribitol) ligase
EC 6.1.1.14 glycine—tRNA ligase
EC 6.1.1.15 proline—tRNA ligase
EC 6.1.1.16 cysteine—tRNA ligase
EC 6.1.1.17 glutamate—tRNA ligase
EC 6.1.1.18 glutamine—tRNA ligase
EC 6.1.1.19 arginine—tRNA ligase
EC 6.1.1.20 phenylalanine—tRNA ligase
EC 6.1.1.21 histidine—tRNA ligase
EC 6.1.1.22 asparagine—tRNA ligase
EC 6.1.1.23 aspartate—tRNAAsn ligase
EC 6.1.1.24 glutamate—tRNAGln ligase
EC 6.1.1.25 lysine—tRNAPyl ligase
EC 6.1.1.26 pyrrolysine—tRNAPyl ligase
EC 6.1.1.27 O-phosphoserine—tRNA ligase
EC 6.1.2 Acid—Alcohol Ligases (ester synthases)
EC 6.1.2.1 D-alanine—(R)-lactate ligase
EC 6.2 Forming Carbon-Sulfur Bonds
EC 6.2.1 Acid-Thiol Ligases
EC 6.2.1.1 acetate—CoA ligase
EC 6.2.1.2 butyrate—CoA ligase
EC 6.2.1.3 long-chain-fatty-acid—CoA ligase
EC 6.2.1.4 succinate—CoA ligase (GDP-forming)
EC 6.2.1.5 succinate—CoA ligase (ADP-forming)
EC 6.2.1.6 glutarate—CoA ligase
EC 6.2.1.7 cholate—CoA ligase
EC 6.2.1.8 oxalate—CoA ligase
EC 6.2.1.9 malate—CoA ligase
EC 6.2.1.10 acid—CoA ligase (GDP-forming)
EC 6.2.1.11 biotin—CoA ligase
EC 6.2.1.12 4-coumarate—CoA ligase
EC 6.2.1.13 acetate—CoA ligase (ADP-forming)
EC 6.2.1.14 6-carboxyhexanoate—CoA ligase
EC 6.2.1.15 arachidonate—CoA ligase
EC 6.2.1.16 acetoacetate—CoA ligase
EC 6.2.1.17 propionate—CoA ligase
EC 6.2.1.18 citrate—CoA ligase
EC 6.2.1.19 long-chain-fatty-acid—luciferin-component ligase
EC 6.2.1.20 long-chain-fatty-acid—[acyl-carrier-protein] ligase
EC 6.2.1.21 covered by EC 6.2.1.30
EC 6.2.1.22 [citrate (pro-3S)-lyase] ligase
EC 6.2.1.23 dicarboxylate—CoA ligase
EC 6.2.1.24 phytanate—CoA ligase
EC 6.2.1.25 benzoate—CoA ligase
EC 6.2.1.26 o-succinylbenzoate—CoA ligase
EC 6.2.1.27 4-hydroxybenzoate—CoA ligase
EC 6.2.1.28 3α,7α-dihydroxy-5β-cholestanate—CoA ligase
EC 6.2.1.29 deleted now EC 6.2.1.7
EC 6.2.1.30 phenylacetate—CoA ligase
EC 6.2.1.31 2-furoate—CoA ligase
EC 6.2.1.32 anthranilate—CoA ligase
EC 6.2.1.33 4-chlorobenzoate—CoA ligase
EC 6.2.1.34 trans-feruloyl-CoA synthase
EC 6.2.1.35 ACP-SH:acetate ligase
EC 6.2.1.36 3-hydroxypropionyl-CoA synthase
EC 6.3 Forming Carbon-Nitrogen Bonds
EC 6.3.1 Acid-Ammonia (or Amine) Ligases (Amide Synthases)
EC 6.3.1.1 aspartate—ammonia ligase
EC 6.3.1.2 glutamate—ammonia ligase
EC 6.3.1.3 now EC 6.3.4.13
EC 6.3.1.4 aspartate—ammonia ligase (ADP-forming)
EC 6.3.1.5 NAD+ synthase
EC 6.3.1.6 glutamate—ethylamine ligase
EC 6.3.1.7 4-methyleneglutamate—ammonia ligase
EC 6.3.1.8 glutathionylspermidine synthase
EC 6.3.1.9 trypanothione synthase
EC 6.3.1.10 adenosylcobinamide-phosphate synthase
EC 6.3.1.11 glutamate—putrescine ligase
EC 6.3.1.12 D-aspartate ligase)
EC 6.3.1.13 L-cysteine:1D-myo-inositol 2-amino-2-deoxy-α-D-glucopyranoside ligase
EC 6.3.1.14 diphthine—ammonia ligase
EC 6.3.2 Acid—Amino-Acid Ligases (Peptide Synthases)
EC 6.3.2.1 pantoate—β-alanine ligase
EC 6.3.2.2 glutamate—cysteine ligase
EC 6.3.2.3 glutathione synthase
EC 6.3.2.4 D-alanine—D-alanine ligase
EC 6.3.2.5 phosphopantothenate—cysteine ligase
EC 6.3.2.6 phosphoribosylaminoimidazolesuccinocarboxamide synthase
EC 6.3.2.7 UDP-N-acetylmuramoyl-L-alanyl-D-glutamate—L-lysine ligase
EC 6.3.2.8 UDP-N-acetylmuramate—L-alanine ligase
EC 6.3.2.9 UDP-N-acetylmuramoylalanine—D-glutamate ligase
EC 6.3.2.10 UDP-N-acetylmuramoylalanyl-tripeptide—D-alanyl-D-alanine ligase
EC 6.3.2.11 carnosine synthase
EC 6.3.2.12 dihydrofolate synthase
EC 6.3.2.13 UDP-N-acetylmuramoyl-L-alanyl-D-glutamate—2,6-diaminopimelate ligase
EC 6.3.2.14 2,3-dihydroxybenzoate—serine ligase
EC 6.3.2.15 deleted, due to EC 6.3.2.10
EC 6.3.2.16 D-alanine—alanyl-poly(glycerolphosphate) ligase
EC 6.3.2.17 tetrahydrofolate synthase
EC 6.3.2.18 γ-glutamylhistamine synthase
EC 6.3.2.19 .................More Read....
Absorbance assays are fast and convenient, since no additional reagents or incubations are required. No protein standard need be prepared. The assay does not consume the protein. The relationship of absorbance to protein concentration is linear. Because different proteins and nucleic acids have widely varying absorption characteristics there may be considerable error, especially for unknowns or protein mixtures. Any non-protein component of the solution that absorbs ultraviolet light will intefere with the assay. Cell and tissue fractionation samples often contain insoluble or colored components that interfere. The most common use for the absorbance assay is to monitor fractions from chromatography columns, or any time a quick estimation is needed and error in protein concentration is not a concern. An absorbance assay is recommended for calibrating bovine serum albumin or other pure protein solutions for use as standards in other methods.
Concentration (mg/ml) = Absorbance at 280 nm divided by path length (cm.)
Pure protein of known absorbance coefficient. Use the following formula for a path length of 1 cm. Concentration is in mg/ml, %, or molarity depending on which type coefficient is used.
concentration = Absorbance at 280 nm divided by absorbance coefficient
To convert units, use these relationships:
Mg protein/ml = % protein divided by 10 = molarity divided by protein molecular weight
Unknowns with possible nucleic acid contamination. Use the following formula to estimate protein concentration:
Concentration (mg/ml) = (1.55 x A280) – 0.76 x A260)
Absorbance coefficients of some common protein standards: