Location via proxy:   [ UP ]  
[Report a bug]   [Manage cookies]                
Scribd Logo
Upload

Welcome to Scribd!

  • 28 views

    Proteins: Laboratory Manual For Practical Exercises Proteins

    Uploaded by

    Sara
    This document provides information about protein precipitation and hydrolysis. It describes how proteins can be reversibly or irreversibly precipitated through various means including changes in pH, temperature, addition of salts or organic solvents. Reversible precipitation is used for protein isolation while irreversible precipitation denatures proteins. The document also outlines an experiment to demonstrate protein hydrolysis using pepsin or hydrochloric acid at different concentrations and temperatures. Materials and procedures are listed to precipitate and hydrolyze egg white proteins as part of practical laboratory exercises.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd

    Proteins: Laboratory Manual For Practical Exercises Proteins

    Uploaded by

    Sara
    28 views5 pages
    This document provides information about protein precipitation and hydrolysis. It describes how proteins can be reversibly or irreversibly precipitated through various means including changes in pH, temperature, addition of salts or organic solvents. Reversible precipitation is used for protein isolation while irreversible precipitation denatures proteins. The document also outlines an experiment to demonstrate protein hydrolysis using pepsin or hydrochloric acid at different concentrations and temperatures. Materials and procedures are listed to precipitate and hydrolyze egg white proteins as part of practical laboratory exercises.

    Original Description:

    .

    Original Title

    proteinss

    Copyright

    © © All Rights Reserved

    Available Formats

    PDF, TXT or read online from Scribd

    Did you find this document useful?

    Is this content inappropriate?

    This document provides information about protein precipitation and hydrolysis. It describes how proteins can be reversibly or irreversibly precipitated through various means including changes in pH, temperature, addition of salts or organic solvents. Reversible precipitation is used for protein isolation while irreversible precipitation denatures proteins. The document also outlines an experiment to demonstrate protein hydrolysis using pepsin or hydrochloric acid at different concentrations and temperatures. Materials and procedures are listed to precipitate and hydrolyze egg white proteins as part of practical laboratory exercises.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    28 views5 pages

    Proteins: Laboratory Manual For Practical Exercises Proteins

    Uploaded by

    Sara
    This document provides information about protein precipitation and hydrolysis. It describes how proteins can be reversibly or irreversibly precipitated through various means including changes in pH, temperature, addition of salts or organic solvents. Reversible precipitation is used for protein isolation while irreversible precipitation denatures proteins. The document also outlines an experiment to demonstrate protein hydrolysis using pepsin or hydrochloric acid at different concentrations and temperatures. Materials and procedures are listed to precipitate and hydrolyze egg white proteins as part of practical laboratory exercises.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    You are on page 1of 5

    Laboratory Manual for Practical Exercises Proteins

    Proteins

    Contents
    Proteins ..............................................................................................................................................1
    Exercise:Reversible precipitation of proteins ...................................................................................2
    Exercise: Irreversible precipitation of proteins .................................................................................3
    Exercise: Hydrolysis of proteins ......................................................................................................5

    Proteins belong between fundamental building and functional components of living systems.
    The proteins with building role are only sparingly soluble in water, the functional ones are mostly
    soluble in water.
    Soluble proteins, like other soluble macromolecules, form colloids. Colloidal dispersion of
    proteins is stabilized by mutual repulsion of corresponding charged particles and mainly by hydrated
    shell. When the hydrated shell is disturbed either by high ionic strength then the solubility of proteins is
    decreased (salting-out). Similar effect happens after addition of lower alcohol or acetone, the hydrated
    shell is removed, the colloidal particles aggregate and are excluded from the solution in the form of
    precipitate. The phenomenon is reversible, especially at low temperature. After dilution with water or
    removing the salts by dialysis precipitated proteins are re-dissolved.
    Besides pH of the environment strongly affects the solubility of proteins. The smallest solubility
    of proteins is at the isoelectric point.

    Protein pI Protein pI

    Pepsin <1,0 Collagen 6,6

    Ovalbumin 4,6 Myoglobin 7,0

    Albumin 4,0 Hemoglobin 7,1

    Tropomyosin 5,1 Ribonuclease 7,8

    Insulin 5,4 Cytochrome c 10,6

    Fibrinogen 5,8 Histone 10,8

    γ-globulin 6,6 Lysozyme 11,0

    1
    Laboratory Manual for Practical Exercises Proteins

    Exercise: Reversible precipitation of proteins


    When proteins lose the electric charge and hydrated shell - aggregate and precipitate from the
    solution. The reversible phenomenon is utilized for isolation of proteins from mixtures. By combination
    of salt concentration and organic solvent can be reached precipitation selectivity of individual protein
    components in a mixture (Cohn’s method of fractionation of blood plasma proteins by ethanol).
    For reversible precipitation of proteins are used salts of alkaline metals and alkaline-earth metals
    (NaCl, Na2SO4, MgSO4), but the most frequently is used ammonium sulphate (NH 4)2SO4. Globulins are
    precipitated at half saturation of the solution with ammonium sulfate, while albumins at full saturation,
    respectively.

    Fig. Protein (in the ring) in aqueous solution (A) and in solution of ammonium sulphate (B).

    Materials
    Test tubes, pipettes.
    Egg white solution (white of one or two eggs is mixed with 1 L of physiological saline), NaCl (solid),
    ammonium sulfate (solid), ethanol, acetone.

    Procedure
    Salting-out of proteins by NaCl and (NH4)2SO4
    1. Add powdered sodium chloride per partes to 1 ml of protein solution in a test tube up to saturation
    of the solution. Proteins are precipitated.
    2. Repeat the procedure with ammonium sulfate instead of sodium chloride.

    Precipitation of proteins by ethanol and acetone


    1. Add a small amount of powdered NaCl to 1 ml of protein solution in a test tube and mix.
    2. Then add several volumes of ethanol per partes during mixing. After a while, flocculation of the
    proteins occurs in the test tube. The precipitate is re-dissolved after addition of water.
    3. Repeat the procedure with acetone instead of ethanol. (Some organic solvents can proteins partly
    denaturated.)

    Observation:

    Conclusions:

    2
    Laboratory Manual for Practical Exercises Proteins

    Exercise: Irreversible precipitation of proteins


    Salts of heavy metals (Pb2+, Hg2+, Cu2+, Ag+) precipitate proteins by direct binding of heavy
    metals to mainly free -SH groups of proteins or by formation of complex species. Therefore the proteins
    (milk) can be used as antidote in the heavy metal intoxication.
    Concentrated mineral acids (for example nitric acid) also precipitate proteins from solutions
    by their dehydration, but mainly by formation of insoluble salts. Precipitation is connected with
    irreversible denaturation of proteins.
    Organic acids precipitate proteins by similar mechanism. In medical practice, precipitation with
    sulfosalicylic acid is used for detection of proteins in urine, whereas trichloroacetic acid is used
    deproteinization (remove of proteins) of biological specimens, as e.g. blood serum.
    Higher temperature - a physical effect, leads to degradation of hydrogen bonds of protein
    molecules, followed by unfolding of polypeptide chains and decreasing of solubility. Almost all proteins
    are precipitated in such a way. Some of them are termolabile, so they are precipitated already at
    temperature 45-50 C. The others are precipitated after shorter or longer boiling. Some proteins found
    in microorganisms living in thermal springs can bear temperatures around 90 C. The proteins are
    denaturated by boiling (especially at the isoelectric point at pH around 5 after acidification of the protein
    solution). In strong acidic and basic solutions proteins are more dissociated, and so they are more
    resistant to higher temperature.

    Materials
    Test tubes, pipettes.
    Lead(II) acetate solution (5 g/l), copper(II) sulfate solution (5 g/l), concentrated HNO3, trichloroacetic
    acid solution (30 g/l), sulfosalicylic acid solution (200 g/l)acetic acid solution (10 g/l), sodium hydroxide
    solution (100 g/l), egg white solution (from the preceding exercise).

    Procedure
    Precipitation of proteins by heavy metal salts
    1. Pipette by 1 ml of protein solution into two test tubes.
    2. Add lead(II) acetate solution dropwise into one of them.
    3. Add copper(II) sulfate solution dropwise into the other one. The proteins are precipitated in both
    test tubes.
    4. After addition of excess of the solutions the precipitates partly re-dissolve. (Why?)

    Precipitation of proteins by mineral acids


    1. Pipette 1 ml of concentrated nitric acid into a test tube.
    2. Pipette carefully 1 ml of protein solution. A white ring of protein precipitate is formed in the
    interface.

    3
    Laboratory Manual for Practical Exercises Proteins

    Precipitation of proteins by organic acids


    1. Pipette by 1 ml of protein solution into two test tubes.
    2. Add few drops of sulfosalicylic acid solution into one of them.
    3. Add few drops of trichloroacetic acid solution into the other one. The white protein precipitates are
    formed in both test tubes.

    Precipitation of proteins by boiling


    1. Pipette by 1 ml of protein solution into three test tubes.
    2. Warm the content of the first one. The proteins are precipitated.
    3. Add a drop of acetic acid solution into the next one and warm up. The proteins are precipitated as
    well.
    4. Add 0.5 ml of sodium hydroxide solution into the last one and warm up again. The proteins are not
    precipitated.

    Observation:

    Conclusions:
    Describe and explain your observation.

    4
    Laboratory Manual for Practical Exercises Proteins

    Exercise: Hydrolysis of proteins


    The peptide bond in protein molecules is relatively stable. It is hydrolyzed in strong acidic
    medium (HCl, 6 mol/L at increased temperature) or in the presence of hydrolytic enzymes, like pepsin
    at pH 1-2 (pH of the gastric juice).

    Materials
    Test tubes, pipettes, a water bath.
    Pepsine solution (2%), concentrated HCl, HCl solution (c = 0.1 mol/L), HCl solution (c = 0.2 mol/L),
    boiled egg white.
    Procedure

    1. Take 4 test tube and number them. Pipette the following substances by the table below:

    Test tube No. 1 2 3 4


    Dist. water (mL) 5 3 - 1
    HCl (0.1 mol/L) (mL) 1 - - -
    HCl (0.2 mol/L) (mL) - - 1 -
    HCl concentrated (mL) - 3 - -
    Pepsin (2 %) (mL) - - 5 5

    2. Add a piece of boiled egg white into each test tube.


    3. Incubate all test tube in a water bath kept at 37 C for 40 min.
    4. Prepare 4 new clean test tubes. Pipette 1 ml of hydrolysate from all test tubes.
    5. Add 2 drops of NaOH (2.5 M) for neutralisation.
    6. Add 1 ml of ninhydrin to all test tubes
    7. Heat in the boiling bath for 5 min.
    8. Record all changes happening in the test tubes. Explain the changes.

    Observation:

    Conclusion:

    You might also like