Lab I: Isolation of Casein from milk and its characterization Flashcards
what is the composition of milk proteins?
casein: 80%
whey: 20%
what are 3 therapeutic effects of casein?
- anticarcinogenic
- decreases plaque and adherence to teeth
- reduces “bad” cholesterol
what are 4 therapeutic effects of whey?
- anticarcinogenic
- helps regulate immune system
- antimicrobial and antiviral
- aids in satiety and weight loss
what is the most prominent phosphoprotein found in milk and cheese?
casein
phosphoproteins definition
proteins that have PHOSPHATE groups attached to amino acid side chains
amino acids definition
organic compounds that serve as the building blocks of proteins
the alpha-C is a ____ Carbon
CHIRAL (the C is bonded to 4 different substituents)
–> most amino acids are OPTICALLY ACTIVE
what is the basic structure of an alpha-amino acid?
the alpha C is bonded to NH2 group, COOH group, H, and R group
which group of the amino acid acts as a base?
which group of the amino acid acts as an acid?
NH2 group (with its lone pair) acts a base
COOH group (acidic proton) acts as an acid
how is a zwitterion formed?
a proton from the COOH group transfers to the NH2 group
the zwitterion is a __ ion
the zwitterion is a DIPOLAR ion since it has both positive and negative charges but is electrically neutral overall (aka with a net charge of 0)
aka the NEUTRAL form of the amino acid
in a zwitterion…
amino group is ___
carboxylic acid group is ___
In a Zwitterion:
Amino group is protonated –> NH3+
Carboxylic acid group is deprotonated –> COO-
what happens when a zwitterion is in BASIC conditions? (aka high pH)
the -OH (base) DEPROTONATES the NH3+ group –> the alpha-C is now bonded to NH2, R, COO-, H
what happens when a zwitterion is in ACIDIC conditions? (aka low pH)
the H+ (acid) PROTONATES the COO- group –> the alpha-C is now bonded to COOH, NH3+, R, H
isoelectric point definition
the pH at which the amino acid exists in its NEUTRAL form (denoted as pI)
- can help us predict if an amino acid will be charged at a specific pH
in a protonated amino acid, how many pKa values do you have and what are they?
2
pKa of carboxyl proton
pKa of NH3+ proton
how do you calculate isoelectric point pI for amino acids? (formula)
pI = (pKa of NH3+ group + pKa of COOH group) / 2
–> aka the average of the two pKa values: pKa of carboxyl proton
pKa of NH3+ proton
xanthoproteic acid test
detects the presence of amino acids with AROMATIC side chains
- heating with conc. HNO3 nitrates the aromatic groups on amino acids to form a yellow/orange colored solution
+ result: solution turns DARK YELLOW/ORANGE
- result: solution does NOT turn dark yellow/orange
how do you perform the xanthoproteic acid test?
heat with conc. HNO3 and treat with a base NaOH –> results in aromatic groups being nitrated
what are the 3 aromatic amino acids?
phenyl alanine
tyrosine
tryptophan
biuret test
identifies the presence of peptide bonds in proteins
- a compound containing at least 2 peptide bonds forms a purple colored solution
+ result: solution turns DEEP PURPLE
–> signifies the presence of proteins
- result: solution stays BLUE
how is a peptide bond formed? what type of reaction is it?
formed between the amino group of one amino acid and the carboxyl group of another (through the loss of a water molecule, namely a CONDENSATION REACTION)
how do you perform the biuret test?
treat with an aq. NaOH solution and aq. CuSO4 solution
benedict’s reagent test (colors)
Detect the presence of reducing sugars in carbohydrates
color of solution:
blue - none
green/yellow - traces of reducing sugar
orange/red - moderate
brick-red - large amounts of reducing sugar
- for milk: the reducing sugar is LACTOSE
what is the benedict’s reagent?
Benedict’s reagent: Mild oxidizing agent composed of Cu (II) sulfate and sodium
citrate in aq. base
what is the cause of the color change in the benedict’s reagent test?
Cu2+ is reduced to Cu1+
what is the reducing sugar in milk?
LACTOSE
reducing sugars defintion
sugars that can act as reducing agents on account of their aldehyde or ketone functional group
reducing sugars are present in __ protein
WHEY protein
sudan III red stain test
detects the presence of LIPIDS
+ result: there is a clear separation between red-fat containing solution and the other immiscible liquid
- result: indicated by HOMOGENOUS red solution
fats are isolated from ___
fats are isolated from CASEIN PROTEIN
sudan III is what type of dye?
a red fat-SOLUBLE dye
what are lipids comprised of?
fatty acids
what are lipids solubility in water vs organic solvents?
lipids are insoluble in water but soluble in organic solvents
what is the pH and pH color of milk?
around 6;
yellow
- you test the pH of milk when you first pour it into the erlenmeyer reaction flask
when and why do you add glacial acetic acid?
when: you add it after the milk has been heated up to 40-45C
why: you add acetic acid to the milk because it denatures the protein; since the isoelectric point of casein is at a lower pH than neutral milk, acid must be added to lower the pH –> when the protein is isoelectric, it is charge NEUTRAL and thus can no longer be soluble in water –> forms a precipitate
what is the pH after you add glacial acetic acid to the reaction flask with milk?
the pH is now orange –> indicating the solution is more ACIDIC than before
what is the purpose of using a cheesecloth?
to separate the whey (liquid) from the casein (solid precipitate)
–> you squeeze the solution with the precipitate and drain out the liquid whey into a beaker labeled whey and then scrape the solid casein into a separate beaker labeled casein
after separating the two types of proteins in milk (aka separating casein and whey), what is the next step?
after separating whey protein and casein protein, we now want to remove the fat from casein
how do we separate the casein from the fats
we add ethanol into the beaker with solid casein (impure still) and then stir with a glass rod, wait for the separation between the 2 layers to form and then DECANT the fats (liquid form) into a separate beaker labeled fats
for the second extraction, add 1:1 diethyl ether and ethanol into the casein beaker and stir again –> then vacuum filtration to get PURE casein protein
what is the filtrate from the vacuum filtration on the casein protein?
a 1:1 ratio of diethyl ether and ethanol
what is the appearance and color of the pure casein protein?
white, grainy precipitate
describe the Biuret test
Biuret test: casein → + result for peptide bond
DI water - deionized water
NaOH
CuSO4 - keep adding until solution turns light PURPLE
describe the xanthoproteic test
Xanthoproteic test: casein → + result for aromatic amino acid
DI water
HNO3
Warm water bath - casein precipitate color turns from white to YELLOW
NaOH - ORANGE color of the fragments
describe the benedict’s reagent test
Benedict’s reagent: whey → + result for presence of reducing sugars
Benedict’s reagent - starts of BLUE
Warm water bath - solution changes from blue to GREEN
describe the sudan III red test
Sudan III red test: fats → + result for the presence of fats
DI water
Sudan III stain - RED ring forms at the top of solution
what type of protein is casein and describe its structure
a phosphoprotein
contains phosphate groups that are attached to some of the amino acid side chains
basically a calcium-phosphate micellar complex
how does casein exist in milk?
casein exists are MICELLES in milk
what are casein micelles composed of?
alpha, beta, and gamma casein
what are micelles?
spherical formation of amphiphilic molecules (aka molecules that have both a hydrophilic part and a hydrophobic part) that forms in a liquid to minimize unfavorable interactions
describe the structure of the casein micelles in milk
hydrophobic inner part: calcium and phosphorus components
hydrophilic outer part
In the casein micelles, the calcium and phosphorus components are enclosed within the spherical structure of the protein, this making the micellar interior hydrophobic, while the outer ends of the micelle are relatively water soluble (hydrophilic).
casein contains which aromatic amino acids?
phenylalanine (Phe)
tyrosine (Tyr)
tryptophan (Trp)
draw the structure of phenylalanine, tyrosine, and tryptophan
what part of the amino acid structure determines the properties of them and the functions the amino acid serves?
the side chain R groups
what makes a molecule optically actice?
if it contains a CHIRAL CENTER - aka a carbon that is bonded to 4 DIFFERENT GROUPS
how do amino acids exist in solution?
amino acids exist as DIPOLAR IONS (AKA ZWITTERIONS)
in the zwitterionic form, the amino group is ___ and the carboxylic group is ___
amino group is PROTONATED (NH3+)
carboxylic group is DEPROTONATED (COO-)
–> this way the positive and negative charges cancel out and the overall molecule is net 0 charge
zwitterion + base reaction
zwitterion + acid reaction
what is the isoelectric point for casein
4.6
what is the purpose of adding glacial acetic acid into milk?
since the isoelectric point for casein is lower than the pH of milk, at the higher pH (of milk) the casein micelles have a net negative charge and are stable
–> by adding glacial acetic acid, the pH of the solution will decrease and cause the casein proteins to precipitate by forming curds and thus can be separated from the liquid whey protein
pH of milk
pH of glacial acetic acid
pH of milk - 6.6
pH of glacial acetic acid - 2.5
how does a dipeptide bond form?
through a condensation/dehydration reaction between the alpha-amino group of one amino acid and the alpha-carboxyl group of another amino acid, with the loss of a water molecule
two amino acids are joined together through a peptide bond (aka amide bond) to form a dipeptide
what is the difference between phenylalanine vs tyrosine and tryptophan in the xanthoproteic test?
phenylalanine does not readily undergo nitration (aka does not get nitrated by NHO3) due to the stability of the phenyl group BUT after prolonged heating, the presence of phenylalanine still shows a positive test (orange/yellow solution)
tyrosine and tryptophan both get undergo nitration by HNO3`
what is lactose composed of
glucose + galactose
what is benedict’s reagent commonly used in?
diabetes tests to identify the presence of reducing sugars (aka sugars that can be oxidized)
lipids definition
a class of organic compounds that are comprised of fatty acids
- characterized by being insoluble in water but soluble in nonpolar organic solvents (ie. chloroform)
what are some examples of common lipids?
oils
waxes
steroids
triglycerides
cholesterol
what functions do lipids serve?
lipids serves as energy reserves
messengers for signaling
components of membranes
which 2 characterization test includes a warm water bath?
xanthoproteic test
benedict’s reagent test
waste disposal protocol:
decanted liquids and filtrate –>
liquids from the 4 tests –>
glassware rinsed with…
decanted liquids and filtrate –> “casein fats, diethyl ether, ethanol and acetone waste” container
liquids from the 4 tests –> “casein test tube waste” container
glassware rinsed with tap water, scrubbed, and poured down sink
which aromatic amino acid was in the picture of the casein structure in the lab handout?
phenylalanine
