• tryptophan

    EC 4 Lyases

    EC 4 Lyases
    EC 4.1 Carbon-Carbon Lyases
    EC 4.1.1 Carboxy-Lyases

    EC 4.1.1.1 pyruvate decarboxylase
    EC 4.1.1.2 oxalate decarboxylase
    EC 4.1.1.3 oxaloacetate decarboxylase
    EC 4.1.1.4 acetoacetate decarboxylase
    EC 4.1.1.5 acetolactate decarboxylase
    EC 4.1.1.6 aconitate decarboxylase
    EC 4.1.1.7 benzoylformate decarboxylase
    EC 4.1.1.8 oxalyl-CoA decarboxylase
    EC 4.1.1.9 malonyl-CoA decarboxylase
    EC 4.1.1.10 deleted, included in EC 4.1.1.12
    EC 4.1.1.11 aspartate 1-decarboxylase
    EC 4.1.1.12 aspartate 4-decarboxylase
    EC 4.1.1.13 deleted
    EC 4.1.1.14 valine decarboxylase
    EC 4.1.1.15 glutamate decarboxylase
    EC 4.1.1.16 hydroxyglutamate decarboxylase
    EC 4.1.1.17 ornithine decarboxylase
    EC 4.1.1.18 lysine decarboxylase
    EC 4.1.1.19 arginine decarboxylase
    EC 4.1.1.20 diaminopimelate decarboxylase
    EC 4.1.1.21 phosphoribosylaminoimidazole carboxylase
    EC 4.1.1.22 histidine decarboxylase
    EC 4.1.1.23 orotidine-5′-phosphate decarboxylase
    EC 4.1.1.24 aminobenzoate decarboxylase
    EC 4.1.1.25 tyrosine decarboxylase
    EC 4.1.1.26 deleted, included in EC 4.1.1.28
    EC 4.1.1.27 deleted, included in EC 4.1.1.28
    EC 4.1.1.28 aromatic-L-amino-acid decarboxylase
    EC 4.1.1.29 sulfoalanine decarboxylase
    EC 4.1.1.30 pantothenoylcysteine decarboxylase
    EC 4.1.1.31 phosphoenolpyruvate carboxylase
    EC 4.1.1.32 phosphoenolpyruvate carboxykinase (GTP)
    EC 4.1.1.33 diphosphomevalonate decarboxylase
    EC 4.1.1.34 dehydro-L-gulonate decarboxylase
    EC 4.1.1.35 UDP-glucuronate decarboxylase
    EC 4.1.1.36 phosphopantothenoylcysteine decarboxylase
    EC 4.1.1.37 uroporphyrinogen decarboxylase
    EC 4.1.1.38 phosphoenolpyruvate carboxykinase (diphosphate)
    EC 4.1.1.39 ribulose-bisphosphate carboxylase
    EC 4.1.1.40 hydroxypyruvate decarboxylase
    EC 4.1.1.41 methylmalonyl-CoA decarboxylase
    EC 4.1.1.42 carnitine decarboxylase
    EC 4.1.1.43 phenylpyruvate decarboxylase
    EC 4.1.1.44 4-carboxymuconolactone decarboxylase
    EC 4.1.1.45 aminocarboxymuconate-semialdehyde decarboxylase
    EC 4.1.1.46 o-pyrocatechuate decarboxylase
    EC 4.1.1.47 tartronate-semialdehyde synthase
    EC 4.1.1.48 indole-3-glycerol-phosphate synthase
    EC 4.1.1.49 phosphoenolpyruvate carboxykinase (ATP)
    EC 4.1.1.50 adenosylmethionine decarboxylase
    EC 4.1.1.51 3-hydroxy-2-methylpyridine-4,5-dicarboxylate 4-decarboxylase
    EC 4.1.1.52 6-methylsalicylate decarboxylase
    EC 4.1.1.53 phenylalanine decarboxylase
    EC 4.1.1.54 dihydroxyfumarate decarboxylase
    EC 4.1.1.55 4,5-dihydroxyphthalate decarboxylase
    EC 4.1.1.56 3-oxolaurate decarboxylase
    EC 4.1.1.57 methionine decarboxylase
    EC 4.1.1.58 orsellinate decarboxylase
    EC 4.1.1.59 gallate decarboxylase
    EC 4.1.1.60 stipitatonate decarboxylase
    EC 4.1.1.61 4-hydroxybenzoate decarboxylase
    EC 4.1.1.62 gentisate decarboxylase
    EC 4.1.1.63 protocatechuate decarboxylase
    EC 4.1.1.64 2,2-dialkylglycine decarboxylase (pyruvate)
    EC 4.1.1.65 phosphatidylserine decarboxylase
    EC 4.1.1.66 uracil-5-carboxylate decarboxylase
    EC 4.1.1.67 UDP-galacturonate decarboxylase
    EC 4.1.1.68 5-oxopent-3-ene-1,2,5-tricarboxylate decarboxylase
    EC 4.1.1.69 3,4-dihydroxyphthalate decarboxylase
    EC 4.1.1.70 glutaconyl-CoA decarboxylase
    EC 4.1.1.71 2-oxoglutarate decarboxylase
    EC 4.1.1.72 branched-chain-2-oxoacid decarboxylase
    EC 4.1.1.73 tartrate decarboxylase
    EC 4.1.1.74 indolepyruvate decarboxylase
    EC 4.1.1.75 5-guanidino-2-oxopentanoate decarboxylase
    EC 4.1.1.76 arylmalonate decarboxylase
    EC 4.1.1.77 4-oxalocrotonate decarboxylase
    EC 4.1.1.78 acetylenedicarboxylate decarboxylase
    EC 4.1.1.79 sulfopyruvate decarboxylase
    EC 4.1.1.80 4-hydroxyphenylpyruvate decarboxylase
    EC 4.1.1.81 threonine-phosphate decarboxylase
    EC 4.1.1.82 phosphonopyruvate decarboxylase
    EC 4.1.1.83 4-hydroxyphenylacetate decarboxylase
    EC 4.1.1.84 D-dopachrome decarboxylase
    EC 4.1.1.85 3-dehydro-L-gulonate-6-phosphate decarboxylase
    EC 4.1.1.86 diaminobutyrate decarboxylase
    EC 4.1.1.87 malonyl-S-ACP decarboxylase
    EC 4.1.1.88 biotin-independent malonate decarboxylase
    EC 4.1.1.89 biotin-dependent malonate decarboxylase
    EC 4.1.1.90 peptidyl-glutamate 4-carboxylase

    EC 4.1.2 Aldehyde-Lyases

    EC 4.1.2.1 deleted, included in EC 4.1.3.16
    EC 4.1.2.2 ketotetrose-phosphate aldolase
    EC 4.1.2.3 deleted
    EC 4.1.2.4 deoxyribose-phosphate aldolase
    EC 4.1.2.5 threonine aldolase
    EC 4.1.2.6 deleted
    EC 4.1.2.7 deleted, included in EC 4.1.2.13
    EC 4.1.2.8 indole-3-glycerol-phosphate lyase
    EC 4.1.2.9 phosphoketolase
    EC 4.1.2.10 (R)-mandelonitrile lyase
    EC .................More Read....

    By admin on 17 Ağustos 2011 | Biomolecules-Biyomoleküller
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    Hartree-Lowry and Modified Lowry Protein Assays

    Hartree-Lowry and Modified Lowry Protein Assays

    Considerations for use

    The Lowry assay (1951) is an often-cited general use protein assay. For some time it was the method of choice for accurate protein determination for cell fractions, chromatography fractions, enzyme preparations, and so on. The bicinchoninic acid (BCA) assay is based on the same princple and can be done in one step, therefore it has been suggested (Stoscheck, 1990) that the 2-step Lowry method is outdated. However, the modified Lowry is done entirely at room temperature. The Hartree version of the Lowry assay, a more recent modification that uses fewer reagents, improves the sensitivity with some proteins, is less likely to be incompatible with some salt solutions, provides a more linear response, and is less likely to become saturated. The Hartree-Lowry assay will be described first.

    Principle

    Under alkaline conditions the divalent copper ion forms a complex with peptide bonds in which it is reduced to a monovalent ion. Monovalent copper ion and the radical groups of tyrosine, tryptophan, and cysteine react with Folin reagent to produce an unstable product that becomes reduced to molybdenum/tungsten blue.

    Equipment

    In addition to standard liquid handling supplies a spectrophotometer with infrared lamp and filter is required. Glass or polystyrene (cheap) cuvettes may be used.

    Procedure – Hartree-Lowry assay

    Reagents

    1. Reagent A consists of 2 gm sodium potassium tartrate x 4 H20, 100 gm sodium carbonate, 500 ml 1N NaOH, H20 to one liter (that is, 7mM Na-K tartrate, 0.81M sodium carbonate, 0.5N NaOH final concentration). Keeps 2 to 3 months.
    2. Reagent B consists of 2 gm 2 gm sodium potassium tartrate x 4 H20, 1 gm copper sulfate (CuSO4 x 5H20), 90 ml H20, 10 ml 1N NaOH (final concentrations 70 mM Na-K tartrate, 40 mM copper sulfate). Keeps 2 to 3 months.
    3. Reagent C consists of 1 vol Folin-Ciocalteau reagent diluted with 15 vols water.

    Assay

    1. Prepare a series of dilutions of 0.3 mg/ml bovine serum albumin in the same buffer containing the unknowns, to give concentrations of 30 to 150 micrograms/ml (0.03 to 0.15 mg/ml).
    2. Add 1.0 ml each dilution of standard, protein-containing unknown, or buffer (for the reference) to 0.90 ml reagent A in separate test tubes and mix.
    3. Incubate the tubes 10 min in a 50 degrees C bath, then cool to room temperature.
    4. Add 0.1 ml reagent B to each tube, mix, incubate 10 min at room temperature.
    5. Rapidly add 3 ml reagent C to each tube, mix, incubate 10 min in the 50 degree bath, and cool to room temperature. Final assay volume is 5 ml.
    6. Measure absorbance at 650 nm in 1 cm cuvettes.

    Analysis

    Prepare a standard curve of absorbance versus micrograms protein (or vice versa), and determine amounts from the curve. Determine concentrations of original samples from the amount protein, volume/sample, and dilution factor, if any.

    Procedure – modified Lowry (room temperature)

    Reagents

    1. Dissolve 20 gm sodium carbonate in 260 ml water, 0.4 gm cupric sulfate (5x hydrated) in 20 ml water, and 0.2 gm sodium potassium tartrate in 20 ml water. Mix all three solutions to prepare the copper reagent.
    2. Prepare 100 ml of a 1% solution (1 gm/100 ml) of sodium dodecyl sulfate (SDS).
    3. Prepare a 1 M solution of NaOH (4 gm/100 ml).
    4. For the 2x Lowry concentrate mix 3 parts copper reagent with 1 part SDS and 1 part NaOH. Solution is stable for 2-3 weeks. Warm the solution to 37 degrees C if a white precipitate forms, and discard if there is a black precipitate. Better, keep the three stock solutions, and mix just before use.
    5. Prepare 0.2 N Folin reagent by mixing 10 ml 2 N Folin reagent with 90 ml water. Kept in an amber bottle, the dilution is stable for several months.

    Assay

    1. Dilute samples to an estimated 0.025-0.25 mg/ml with buffer. If the concentration can’t be estimated it is advisable to prepare a range of 2-3 dilutions spanning an order of magnitude. Prepare 400 microliters each dilution. Duplicate or triplicate samples are recommended.
    2. Prepare a reference of 400 microliters buffer. Prepare standards from 0.25 mg/ml bovine serum albumin by adding 40-400 microliters to 13 x 100 mm tubes + buffer to bring volume to 400 microliters/tube.
    3. Add 400 microliters of 2x Lowry concentrate, mix thoroughly, incubate at room temp. 10 min.
    4. Add 200 microliters 0.2 N Folin reagent very quickly, and vortex immediately. Complete mixing of the reagent must be accomplished quickly to avoid .................More Read....

    By admin on 15 Temmuz 2011 | Biochemistry Protocols - Biyokimya Protokoller, Proteins- Proteinler
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    Bradford protein assay

    Bradford protein assay

    Considerations for use

    The Bradford assay is very fast and uses about the same amount of protein as the Lowry assay. It is fairly accurate and samples that are out of range can be retested within minutes. The Bradford is recommended for general use, especially for determining protein content of cell fractions and assesing protein concentrations for gel electrophoresis.

    Assay materials including color reagent, protein standard, and instruction booklet are available from Bio-Rad Corporation. The method described below is for a 100 µl sample volume using 5 ml color reagent. It is sensitive to about 5 to 200 micrograms protein, depending on the dye quality. In assays using 5 ml color reagent prepared in lab, the sensitive range is closer to 5 to 100 µg protein. Scale down the volume for the “microassay procedure,” which uses 1 ml cuvettes. Protocols, including use of microtiter plates are described in the flyer that comes with the Bio-Rad kit.

    Principle

    The assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color change. The assay is useful since the extinction coefficient of a dye-albumin complex solution is constant over a 10-fold concentration range.

    Equipment

    In addition to standard liquid handling supplies a visible light spectrophotometer is needed, with maximum transmission in the region of 595 nm, on the border of the visible spectrum (no special lamp or filter usually needed). Glass or polystyrene (cheap) cuvettes may be used, however the color reagent stains both. Disposable cuvettes are recommended.

    Procedure

    Reagents

    1. Bradford reagent: Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml 95% ethanol, add 100 ml 85% (w/v) phosphoric acid. Dilute to 1 liter when the dye has completely dissolved, and filter through Whatman #1 paper just before use.
    2. (Optional) 1 M NaOH (to be used if samples are not readily soluble in the color reagent).

    The Bradford reagent should be a light brown in color. Filtration may have to be repeated to rid the reagent of blue components. The Bio-Rad concentrate is expensive, but the lots of dye used have apparently been screened for maximum effectiveness. “Homemade” reagent works quite well but is usually not as sensitive as the Bio-Rad product.

    Assay

    1. Warm up the spectrophotometer before use.
    2. Dilute unknowns if necessary to obtain between 5 and 100 µg protein in at least one assay tube containing 100 µl sample
    3. If desirred, add an equal volume of 1 M NaOH to each sample and vortex (see Comments below). Add NaOH to standards as well if this option is used.
    4. Prepare standards containing a range of 5 to 100 micrograms protein (albumin or gamma globulin are recommended) in 100 µl volume. See how to set up an assay for suggestions as to setting up the standards.
    5. Add 5 ml dye reagent and incubate 5 min.
    6. Measure the absorbance at 595 nm.

    Analysis

    Prepare a standard curve of absorbance versus micrograms protein and determine amounts from the curve. Determine concentrations of original samples from the amount protein, volume/sample, and dilution factor, if any.

    Comments

    The dye reagent reacts primarily with arginine residues and less so with histidine, lysine, tyrosine, tryptophan, and phenylalanine residues. Obviously, the assay is less accurate for basic or acidic proteins. The Bradford assay is rather sensitive to bovine serum albumin, more so than “average” proteins, by about a factor of two. Immunoglogin G (IgG – gamma globulin) is the preferred protein standard. The addition of 1 M NaOH was suggested by Stoscheck (1990) to allow the solubilization of membrane proteins and reduce the protein-to-protein variation in color yield.

    References

    • Bradford, MM. A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254. 1976.
    • Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990).

    .................More Read....

    By admin on | Biochemistry Protocols - Biyokimya Protokoller, Proteins- Proteinler
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