REVIEWER: BIOCHEMISTRY LAB

ISOLATION AND ELEMENTARY COMPOSITION OF PROTEINS

What are PROTEINS?

 Proteins are macromolecules
 Constructed from one or more unbranched chains of amino
acids; they are polymers.
 Typical protein contains 200-300 amino acids but some are
much smaller (smallest are called peptides) and some much
larger (titin; a protein found in skeletal and cardiac
muscle; contains 26,926 amino acids in a single chain)

GENERAL STUCTURE OF AN -AMINO ACID

SAMPLE: WHOLE MILK

 COMPOSITION
 Vitamins (thiamine, riboflavin, panthothenic acid and
vitamins A, B12 and D)
 Minerals (Ca, Na,P, K, and trace minerals)
 Proteins
 Carbohydrates (mostly lactose)
 Lipids (fats)

PROTEINS IN MILK:
 Caseins
  Lactalbumins
 Lactoglobulins

WHAT IS CASEIN?
 A protein that is found in milk and used independently in
many foods as a binding agent.
 It is part of a group called phosphoproteins, collections of
proteins bound to something containing phosphoric acid.
SYNONYMS
 Milk solids
Description
 Calcium caseinate is produced from skim milk by adding an
acid to cause a protein to coagulate, where it can be
filtered to separate the curds from the whey.
 Sodium caseinate is produced by reacting the acid casein with
sodium hydroxide.

CASEINS
 The casein content of milk represents about 80% of milk
proteins.
 The principal casein fractions are alpha(s1) and alpha(s2)-
caseins,ß-casein, and kappa-casein.

CASEINS: PHOSPHOPROTEINS
 CHEMICAL FORMULA:
 Casein proteins are composed of the following amino acids:

Glutamic acid 20.2% Aspartic acid 6.4% Threonine 4.4% Glycine 2.4%

Proline 10.2% Serine 5.7% Arginine 3.7% Tryptophan 1.1%

Leucine 8.3% Tyrosine 5.7% Histidine 2.8% Cystine 0.3%

Lysine 7.4% Isoleucine 5.5% Alanine 2.7%

Valine 6.5% Phenylalanine 4.5% Methionine 2.5%

 The conformation of caseins is much like that of denatured

globular proteins.
 The high number of proline residues in caseins causes
particular bending of the protein chain and inhibits the
formation of close-packed, ordered secondary structures.
 Caseins contain no disulfide bonds. As well, the lack of
tertiary structure accounts for the stability of caseins
against heat denaturation because there is very little
structure to unfold.
 Without the tertiary structure there is considerable exposure
of hydrophobic residues. This results in strong association
reactions of the caseins and renders them insoluble in water.
USES:
 Caseinates are used as sources of protein for nutrition.
 They are good emulsifiers, helping fats to stay suspended in
water-based products such as milkshakes, coffee creamers, and
ice creams.
 They are used as binders in processed meats (luncheon meat,
sausages, etc.)
 Caseins are used in wine-making to clarify the wine, by
causing fine particles to coagulate with the protein so they
can be easily filtered out or precipitated.
  Caseins are used as food colors, where they make a nice
opaque white color, which can then be tinted with other
colors as required.
 Casein proteins were one of the first plastics, and they are
still in use for this purpose. Thin plastic films of casein
can be made by adding glycerol or sorbitol as a plasticizer,
a substance that lowers the temperature at which a plastic
softens and makes it more pliable.

ISOLATION

 Mongrel ion or hybrid ion

 Isoelectric point- the pH where the zwitterion is formed

ISOELECTRIC ISOLATION
 pH or Calcium caseinate = 4.6
- insoluble in solutions with a pH less than 4.6
 The pH of milk is 6.6, therefore casein has a negative charge
at this pH and is solubilized as a salt.
 If an acid is added to milk, the negative charges on the
outer surface of the casein micelles are neutralized, by
protonation of the phosphate groups.
 Casein micelles are destabilized or aggregate because the
electric charge is decreased to that of the isoelectric point
(pH at which there is no net charge because there are equal
number of positive and negative charges present).
 Casein micelles disintegrate and the casein (the neutral
protein) precipitates because it is no longer polar, with the
calcium ions remaining in solution.
 The milk is heated to 40°C (optimal temp for denaturation)
- Formation of curds
 Acetic acid is added, drop by drop
- to adjust the pH to the isoelectric point of casein.
- This will cause the casein to “clot” and precipitate out
along with butterfat leaving a liquid component called
whey.
- the liquid will change from milky to almost clear when
more casein separates.
 Heating the milk causes the micelles to dissociate more
readily when the pH is lowered, freeing up the amino acids.
- It is important to not heat the milk to hot because over
the optimal temperature the curds dissociate quickly into
fine particles and are no longer curds.
 If too much acetic is added.
- The protein precipitate will dissolve.
 The casein and butterfat are separated from the whey by
straining the precipitate through cheesecloth.
 Casein is insoluble in ethanol, so this property is used to
remove the unwanted fat from the preparation.
 The casein is then dried. The curds will need to be broken up
by mashing to remove as much liquid as possible.
ELEMENTARY COMPOSITIONS

TEST FOR CARBON, HYDROGEN, OXYGEN

 Decomposition by combustion (incomplete)

TEST FOR NITROGEN

 Reaction of casein with soda lime (NaOH) hydrolyzes the
protein to amino acids and then deaminated by OH- (catalyst)
to give off NH3

SODIUM FUSION
 Decomposition of organic-bound elements (e.g. N, S, P,
halogens, etc.) by converting into their ionic species (as
sodium salts)
TEST FOR SULFER (as sulfate ion)

 Precipitation of barium sulfate in acidic pH using BaCl2

solution.

TEST FOR PHOSPHORUS (as phosphate ion)

 Precipitation of ammonium phosphomolybdate in acidic medium

COLOR REACTIONS OF PROTEINS AND AMINO ACIDS

TYPES OF REACTIONS UNDERGONE BY PROTEINS

1. Reactions due to the colloidal nature of proteins
- Heat coagulation, salting out.
2. Chemical reaction between protein and the reagent (due to
acidity and basicity of protein)
- Precipitation by mineral acids, salts of heavy metals,
alkaloidal reagents
3. Reactions due to presence of chemical groups in the protein
NINHYDRIN REACTION

 Ninhydrin (triketohydrindenehydrate) reacts with ⍺-amino

acids to give intense blue or purple-colored compound

BIURET TEST

 Compound containing two -CONH2 joined either directly together

or through a N or C atom
 Reaction with alkaline CuSO4 giving colored coordination
complex
  The intensity of the color is dependent on the no. of peptide
bonds present, the more peptide bonds the darker is the
color.

XANTHROPROTEIC TEST

 Involves nitration of phenyl group upon heating with conc.

HNO3

 Yellow nitro derivatives or orange-colored salts of

derivatives are formed under alkaline conditions

MILLON-NASSE TEST

 Reaction of the hydroxylphenyl group with the reagent (HgSO4

in H2SO4) forming a colored old rose complex (probably the
mercuric salt of the phenolic compound)
 Positive result rendered by any phenolic compound with
unsubstituted 3,5 positions, e.g. tyrosine
HOPKINS-COLE REACTION

 Indole group (like in tryptophan) condensed with aldehyde

(e.g. glyoxylic acid) in cone. H2SO4 to form a violet complex

BROMINE WATER TEST

 Addition of bromine water and n-amyl alcohol to a sample with

tryptophan results in formation of pinkish lavender or violet
color in the alcohol layer
PAULY REACTION

 Sulfanilic acid with nitrous acid (HONO) form diazobenzene

sulfanoic acid by diazotization

 Diazobenzene SA couples with amines, phenols and imidazoles

to form highly-colored azo dyes (mono- and bis-azo products);
bis-azo products preferred or predominate at higher pH
LEAD ACETATE REACTION

 Strong alkali splits off a part of protein sulfur (loosely

combined sulfer) like in Cysteine and Cystine as sulfides(S2-)
which are precipitated as black PbS.

SAKAGUCHI REACTION

 Substances containing the guanidino group,NH2C(=NH)NH, react

with ⍺-naphthol and oxidizing agent (NaOBr) to give a red or
orange color