Isolation of Casein Flashcards
What is milk composed of? State the percentages.
Case - 80%
Whey - 20%
What are the therapeutic effects of casein?
- Anticarcinogenic
- Decreases plaque and adherence to teeth
- Reduces bad cholesterol
What are the therapeutic effects of whey?
- Anticarcinogenic
- Helps regulate immune system
- Antimicrobial and antiviral
- Aids in Satiety and weight loss
Casein is the most prominent phosphoprotein found in milk and cheese. What are phosphoproteins?
Have phosphate groups attached to amino acid side chains
What are amino acids? What is the basic alpha-amino acid structure?
Amino acids are organic compounds that serve as the building blocks of proteins.
𝛼-amino
acids and are characterized by a central carbon (termed as the 𝛼𝛼-carbon) that is bonded to 4
different functional groups: a hydrogen atom, an amino group (-NH2 ), a carboxylic acid group (-
COOH), and a side chain “R” group.
Basic alpha-amino acid structure drawn in notes
Is the alpha carbon in amino acids chiral or chiral? Does this mean amino acids are optically active or optically inactive?
Chiral
Optically acitve
Since the 𝛼𝛼-carbon is bonded to four different groups, the
𝛼-amino acids are optically active.
What is the xanthoproteic acid test?
Xantho means yellow
This test detects the presence of amino acids(proteins) with aromatic side chains, such as tyrosine, tryptophan, and phenyl alanine. To do the test, you need to add HNO2, warm, cool and then add NaOH.
Positive test: the solution turn dark yellow/orange
Negative result: the solution does not turn the solution dark yellow/orange
What is the isoelectric point? How is it calculated?
The isoelectric point is the pH at which the amino acid exists in its neutral form (i.e. the positive charges are balanced but eh negative charges on the amino acid so that the net charge on the amino acid is zero). It is denoted as pI.
In a protonated amino acid, you have two pKa’s: The pKa of carboxyl proton and the pKa of NH3 proton.
For amino acids, we can calculate pI by averaging the above two pKa values
pI = (pKa of NH3 group) + (pKa of COOH group) / 2
What is the biuret test?
This test identifies the presence of peptide bonds in proteins. In this test, you need to treat the casein with an aqueous NaOH solution and aqueous CuSO4 solution.
Positive result turns the solution a deep purple. A negative result keeps the solution blue.
What is a peptide bond?
Formed between the alpha-amino group of one amino acid and the alpha carboxyl group of another (through the loss of a water molecule, namely a condensation /dehydration reaction). Essentially, two amino acids are joined together by a peptide bond (or an amide bond) to form a dipeptide.
What is Benedict’s reagent test?
Detects the presence of reducing sugars in carbohydrates. The reducing sugar in milk is lactose(which is made up of glucose and galactose). These are present in the whey protein. Thus, this test looks for sugars present in the whey protein.
The reagent itself is composed of a mild oxidizing agent composed of Cu (II) sulfate and sodium citrate in aqueous base. It is added to the test tube and warmed.
The change in color that occur as a result happen because cu2+ is reduced to cu+.
Blue homogenous solution Indicates that there are no reducing sugars. Then it’s green, then yellow. Red indicates a large amount of reducing sugars.
What are reducing sugars?
Sugars that can act as reducing agents on account of their aldehyde or ketone functional group
What is the Sudan III Red stain test?
Sudan III is a red fat-soluble dye that detects the presence of lipids(i.e. fats). The lipids are comprised of fatty acids - insoluble in water but soluble in non-polar organic solvents.
To complete this test, the stain is added along with DI H2O.
Sudan II red dye is added to the fats isolated from casein protein. A positive result is indicated by a red halo(i.e. a clear separation between red-fat containing solution at the top of the test tube and the other immiscible liquid that will be a cloudy white layer at the bottom of the test tube). A negative result is indicated by a homogenous red solution.
What parts of the amino acid act as the base and which act as the acid?
NH2 group (with its lone pair) acts as a base
COOH group(acidic proton) acts as an acid
In solution, amino acids exist as dipolar ions, referred to as zwitterions. Draw the zwitterion(dipolar ion) and explain how it is made.
Refer to notes
The transfer of a proton from the COOH to the NH2 group from a zwitterion.
In a zwitterion, there are both positive and negative charges, but overall
It is electrically neutral with a net charge of zero
Draw the pH related forms of amino acids on the zwitterion
refer to notes
Draw the flow chart for this experiment
Refer to notes
What is the waste disposal for this lab?
Liquids decanted and filtrates should be disposed of into the “Casein fats, diethyl ether, ethanol and acetone waste”
Liquid from all tests should be disposed of in the “Casein test tube waste”
All glassware should be rinsed with tap water, scrubbed with the glassware brush, and poured down the sink.
Describe the general procedure
- Add milk to an Erl. Flask
- Warm the milk in a warm water bath.
- Add glacial acetic acid while flask is still in warm water bath
- After all the acetic acid has been added, let the beaker cool for 5 mins
- Isolate the casein protein using a cheesecloth. Keep the extracted liquid which is whey
- To remove the fat from the casein: Add ethanol to the casein, stir, and let the solid settle. The fat will dissolve in the alcohol. Decant the liquid into a beaker labeled “fats”. The left-over solid casting should have diethyl ether and ethanol added to it and the solid collected by vacuum filtration.
In milk, casein
exists as micelles. Casein micelles are composed of (1). In the casein
micelles, the (2) and (3) components are enclosed within the spherical structure of
the protein, this making the micellar interior (4), while the outer ends of the micelle are (5)
- alpha, beta, and gamma casein
- Calcium
- Phosphorus
- Hydrophobic
- relatively water soluble (hydrophilic).
What part of the amino acid determines its properties and the functions they serve?
The side chains (R groups) determine the properties of amino
acids and the functions they serve.
At what pH does the isoelectric point occur for casein?
4.6
The pH of milk is (1). At this pH, casein micelles have a net (2) charge and are quite stable. Casein is precipitated
from milk by acidifying it.
(1) 6.6
(2) negative
