EXP 3: PRECIPITATION OF PROTEINS

1. PPT by Heat Coagulation

2. PPT by Organic Solvents

3. PPT by Salting Out

4. PPT by Heavy Metal Ions

5. PPT by Alkaloidal Reagents

What are the tests involved in Experiment 3?

Fake, electric charge.

Real or Fake? The solubility of proteins from colloidal systems depends on the isoelectric point and the layer of hydration around them.

Precipitation by Heat Coagulation

What is the first test in Experiment 3?

Isoelectric Point

Heat Coagulation and Precipitation occurs best near the ___ of the protein.

Real

Real or Fake? Casein proteins are denatured by a change of pH.

Heat

This disrupted hydrophobic interactions and hydrogen bonds by making molecules vibrate too violently. This produces coagulation, as in frying an egg.

Aqueous Solutions

These are not coagulated by heat. Proteases, Peptones, Gelatin and Casein are some examples of these solutions.

Real

Real or Fake? Casein is not denatured in heat.

Casein and Albumin Solution

What are the names of the reactants for the PPT by Heat Coagulation?

C81H125N22O39P and C123H193N35O37

What are the chemical formulas for the reagents used in PPT by Heat Coagulation?

C81H125N22O39P

What is the chemical formula of Casein?

C123H193N35O37

What is the chemical formula of albumin?

1N Acetic Acid (CH3COOH) and 5N Acetic Acid (CH3COOH)

What are the reagents for PPT by Heat Coagulation?

1N Acetic Acid (CH3COOH)

What reagent yielded the best results in coagulation?

Denaturation

What is the process wherein proteins or nucleic acids lose quaternary, tertiary, and secondary structure because of the presence of stress, which may be caused by acids, bases, salts, or heat?

Acetic acid

What is the most important of the carboxylic acids which is also called ethanoic acid?

Real

Real or Fake? Heat causes the denaturation of proteins by increasing the kinetic energy of the molecules and by breaking weak bonds like electrostatic interactions, hydrogen bonds, and hydrophobic interactions.

electrostatic interactions, hydrogen bonds, and hydrophobic interactions

Give three examples of weak bonds broken down by heat in the denaturation process.

Fake, insoluble

Real or Fake? The denatured protein becomes soluble and precipitates out.

dilute acetic acid

What provides the acidic medium that brings the pH close to its isoelectric pH?

Fake, minimum

Real or Fake? At Isoelectric pH, the protein has a maximum solubility and is thus precipitated.

Normality

What is the measure of concentration equal to the gram equivalent weight per liter of solution? This is also known as the equivalent concentration of a solution.

Precipitation by Organic Solvents

What is the second test in Experiment 3?

Ethyl Alcohol (CH3CH2OH)

What is the organic solvent used in the second test in Experiment 3?

CH3CH2OH

What is the chemical formula of the solvent used in the PPT by Organic Solvent?

Dehydration

What is the major process that occurs in the PPT by organic solvents?

2% Albumin

What is the reactant for the Precipitation by Organic Solvents?

Fake, insoluble

Real or Fake? Organic solvents precipitate proteins, and when made to stand, sometimes they are denatured and become soluble in water.

Acetone

What is another organic solvent like ethanol that can also precipitate proteins by reducing the amount of water and by reducing the dielectric constant?

lower dielectric constants

Why are water-miscible organic solvents such as acetone and ethanol generally good protein precipitants?

acetone and ethanol

Water-miscible organic solvents, such as ___ and ___ are generally good protein precipitants because their lower dielectric constants lower the solvating power of their aqueous solutions for dissolved proteins.

precipitating membrane proteins

Alcohol rubbed over the injured site acts by p___ m___ p___ of the microorganisms, and hence the multiplication of the microorganisms is prevented at the site of tissue injury.

Alcohol

What functions well in the inhibition of the growth and reproduction of many microorganisms, including bacteria, fungi, protozoa, and viruses?

Real

Real or Fake? Human skin cells are more resistant to alcoholic coagulation than most microorganisms. This is why the human skin doesn't coagulate if it comes into contact with alcohol.

Hardens the tissues for preservation purposes.

how is the process applied in the fixing of tissue for histological purposes?

white

What is color is the precipitate when you combine albumin with ethanol?

Precipitation by Salting Out

What is the third test in Experiment 3?

red ppt

What indicates a positive result with protein in the PPT by salting out by Millon’s reaction?

Ammonium Sulfate

What is added to albumin and peptone to make it saturated?

Blue

What color indicates a negative in the biuret’s test indicating all proteins have been salte out?

Real

Real or Fake? A positive reaction means that proteins haven’t been fully salted out by ammonium sulfate and the separation is incomplete, yielding a violet color.

Millon’s Reaction and Biuret Reaction

What are the tests used to test for the precipitate in the Precipitation by Salting Out?

Precipitation of Proteins

What does the Millon’s Reaction verify in the Precipitation by Salting Out Test?

Millon’s Reagent (HgN2O6)

What is the neutral salt that precipitated the albumin in Exp. 3?

Biuret Reaction

What test verified the absence of proteins in the solution?

Solid Ammonium Sulfate (NH4)SO4

What was added to 2% Albumin to saturate it for the Precipitation of Salting Out Test?

Pink

What color is peptone in biuret’s test?

in the filtrate

After filtering the solution of peptone and ammonium sulfate, where is the peptone found?

Real

Real or Fake? The addition of salts and electrolytes leads to the absorption of the solution envelope from the colloidal particles of a protein solution, along with the neutralization of surface charges leading the protein to precipitate.

Salting out

What is a purification method that utilizes the reduced solubility of molecules in a solution of high ionic strength and causes a reduction of the molecule’s solubility of hydrophilic groups/charged amino acids, which requires water molecules to surround them to remain dissolved?

Real

Real or Fake? In an aqueous solution with high ionic strength, the water molecules surround the ions and proteins. At a certain ionic strength, the water molecules can’t support the charges of both ions and the proteins

Hoffmeister-Serie

What series induces selective precipitation?

Precipitation by Heavy Metal Ions

What is the fourth test in Experiment 3?

2% Albumin (C123H193N37O35)

What is the reactant in the PPT by Heavy Metal Ions?

Lead Acetate [Pb(Ac)2] - Silver Nitrate 1% (AgNO3) - Copper Sulfate 1% (CuSO4)

What are the reagents used in the PPT by Heavy Metal Ions?

Egg Albumin

This is used as an antidote for mercury or lead poisoning due its property to be coagulated by metallic ions.

Lead Acetate (Pb (Ac)2) and Silver Nitrate (AgNO3)

what reagents yielded a white precipitate before the addition of NaOH?

Copper Sulfate (CuSO4)

What yielded a blue solution in