• spectrophotometer

    AMINO ACID ASSAY

    AMINO ACID ASSAY

    BY NINHYDRIN COLORIMETRIC METHOD

    Prepared by

    Nam Sun Wang

    Department of Chemical & Biomolecular Engineering

    University of Maryland

    College Park, MD 20742-2111

    ENCH485

    Method

    The reaction between alpha-amino acid and ninhydrin involved in the development of color are described by the following five mechanistic steps:

      alpha-amino acid + ninhydrin ---> reduced ninhydrin + alpha-amino acid + H2O

  alpha-amino acid + H2O ---> alpha-keto acid +NH3

  alpha-keto acid + NH3 ---> aldehyde + CO2

    Step (1) is an oxidative deamination reaction that removes two hydrogen from the alpha-amino acid to yield an alpha-imino acid. Simultaneously, the original ninhydrin is reduced and loses an oxygen atom with the formation of a water molecule. In Step (2), the NH group in the alpha-imino acid is rapidly hydrolyzed to form an alpha-keto acid with the production of an ammonia molecule. This alpha-keto acid further undergoes decarboxylation reaction of Step (3) under a heated condition to form an aldehyde that has one less carbon atom than the original amino acid. A carbon dioxide molecule is produced here. These first three steps produce the reduced ninhydrin and ammonia that are required for the production of color in the last two Steps (4) and (5). The overall reaction for the above reactions is simply (slightly inaccurately) expressed in Reaction (6) as follows:

    alpha-amino acid + 2 ninhydrin ---> CO2 + aldehyde + final complex(BlUE) + 3H2O

    In summary, ninhydrin, which is originally yellow, reacts with amino acid and turns deep purple. It is this purple color that is detected in this method.Ninhydrin will react with a free alpha-amino group, NH2-C-COOH. This group is contained in all amino acids, peptides, or proteins. Whereas, the decarboxylation reaction will proceed for a free amino acid, it will not happen for peptides and proteins. Thus, theoretically only amino acids will lead to the color development. However, one should always check out the possible interference from peptides and proteins by performing blank tests especially when such solutions are readily available. For example, one can simply add the ninhydrin reagent to a solution of only proteins and see if there is any color development. There is no excuse for failing to perform such a vital test when the sample mixture contains both proteins and amino acids. There are also reports that chemical compounds other than amino acids also yield positive results.

    This test can be used routinely for the detection of glycine in the absence of other interfering species. Although this is a fast and sensitive test for the presence of alpha-amino acids, because of the nonselectivity, it cannot be used to analyze the relative individual contents of a mixture of different amino acids. Furthermore, the color intensity developed is dependent on the type of amino acid. Finally, it does not react with tertiary or aromatic amines.

    Note that since ninhydrin is a strong oxidizing agent, proper caution should be exercised in handling this compound. It is especially potent at the elevated temperature under which the reaction is carried out. The ninhydrin reagent will stain the skin blue and cannot be immediately washed off completely if it comes in contact with the skin. However, as in any other stain on the skin, the color will gradually rub off after about a day.

    List of Reagents and Instruments

    A. Equipment

      Test tubes
      Pipets
      Spectrophotometer

    B. Reagents

      Ninhydrin Reagent Solution

        Ninhydrin: 0.35 g
        Add ethanol to: 100 ml (See Note 1.)

    Procedures

      Add 1 ml of the ninhydrin solution to 5 ml of sample. Cover the test tube with a piece of paraffin film to avoid the loss of solvent due to evaporation. A capped test tube can also be used instead.
      With gentle stirring, react at 80-100ºC for 4-7 minutes. (How would one find out the amount of time needed to ensure a complete reaction?) If a large heated water bath is used for the entire class and if there is no good provision for holding the test tube in the hot water bath, the test tube may be held with a piece of wire and hang on the side of the water container. A clamp usually does not work too well.
      After cooling to room temperature in a cold water bath, record the absorbance with       .................More Read....

    By admin on 19 Temmuz 2011 | Biochemistry Protocols - Biyokimya Protokoller, Experimental Biochemistry - Deneysel Biyokimya
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    Absorbance Assay (280 nm)

    Absorbance Assay (280 nm)

    Considerations for use

    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.

    Principle

    Proteins in solution absorb ultraviolet light with absorbance maxima at 280 and 200 nm. Amino acids with aromatic rings are the primary reason for the absorbance peak at 280 nm. Peptide bonds are primarily responsible for the peak at 200 nm. Secondary, tertiary, and quaternary structure all affect absorbance, therefore factors such as pH, ionic strength, etc. can alter the absorbance spectrum.

    Equipment

    In addition to standard liquid handling supplies a spectrophotometer with UV lamp and quartz cuvette are required.

    Procedure

    Carry out steps 1-4 (280 nm only) for a very rough estimate. Carry out all steps if nucleic acid contamination is likely.
    1. Warm up the UV lamp (about 15 min.)
    2. Adjust wavelength to 280 nm
    3. Calibrate to zero absorbance with buffer solution only
    4. Measure absorbance of the protein solution
    5. Adjust wavelength to 260 nm
    6. Calibrate to zero absorbance with buffer solution only
    7. Measure absorbance of the protein solution

    Analysis

    Unknown proteins or protein mixtures. Use the following formula to roughly estimate protein concentration. Path length for most spectrometers is 1 cm.

    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)

    Comments

    Cold solutions can fog up the cuvette, while warm solutions can release bubbles and interfere with the readings. For concentrated solutions (absorbance greater than 2) simply dilute the solution.

    Absorbance coefficients of some common protein standards:

    • Bovine serum albumin (BSA): 63
    • Bovine, human, or rabbit IgG: 138
    • Chicken ovalbumin: 70

    References

    • Layne, E. Spectrophotometric and Turbidimetric Methods for Measuring Proteins. Methods in Enzymology 3: 447-455. 1957.
    • Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69. 1990.
    .................More Read....

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