Characterization
of Proteins
Amino Acids
Amino Acids
Amino Acids
Accdg. To Biological
Function
1. CATALYST
2. TRANSPORT PROTEINS
3. NUTRIENT and STORAGE PROTEINS
4. STRUCTURAL PROTEINS
5. CONTRACTILE and MOTILE PROTEINS
6. DEFENSE PROTEINS
7. REGULATORY PROTEINS
Accdg. to Shape
1. GLOBULAR PROTEINS
- polypeptide chain/s folded into
spherical or globular shape
-soluble in aq. system
-ex. enzymes
2. FIBROUS PROTEINS
- polypeptide chains arranged in long
strands or sheets
-water insoluble
-ex, keratin, collagen, fibroin
Accdg. To Solubility
1. ALBUMINS
- soluble in water and dilute aq. solutions
2. GLOBULINS
- soluble in dilute salt solutions but are insoluble or
sparingly soluble in water
3. GLUTELINS
- soluble in dilute solutions of acids and bases
- insoluble in neutral solvents
4. PROLAMINS
- soluble in 50-90% alcohol
- insoluble in water, neutral solvents or absolute
alcohol
5. ALBUMINOIDS/SCLEROPROTEINS
- insoluble in in most ordinary solvents
Protein Denaturation
A loss of three-dimensional structure sufficient to
cause of loss of function.
Types:
1. Irreversible Denaturation
- biological function /activity cannot be
regained
2. Reversible Denaturation
- biological function/activity can be regained
Denaturating Agents
Proteins can be denatured by:
a. Strong acids and bases
b. Organic solvents
c. Detergents
d. Reducing agents
e. Salts
Salting-in
Salting-out
f. Heavy metals
g. Temperature
Protein Hydrolysis
1. COMPLETE HYDROLYSIS
- uses a strong acid or base + high T
- product/s: amino acids
2. INCOMPLETE/PARTIAL HYDROLYSIS
- uses enzymes called protease
- product/s: mixture of amino acids and
oligopeptides
COMPLETE HYDROLYSIS
1. ACID HYDROLYSIS
- most commonly used reagent is 6N HCl
- disadvantages:
a. partial destruction of cys and tyr
b. complete destruction of trp
c. incomplete liberation of val and ile
d. racemization and destruction of ser and thr
e. asn + gln converted to asp + glu
COMPLETE HYDROLYSIS
2. ALKALINE HYDROLYSIS
- uses NaOH or KOH
-advantages:
a. trp not destroyed
-disadvantages:
a. arg, asn, gln, ser are
destroyed
INCOMPLETE HYDROLYSIS
-specific peptide bonds hydrolyzed by
proteases like:
Trypsin
Chymotrypsin
Pepsin
Bromelain
Papain
Separation/Purification of Proteins
Properties of proteins being considered:
1. Charge
2. Molecular size, shape
3. Solubility
4. Affinity to a ligand
5. pI
Casein
- Phosphoprotein (Phosphate groups
attached to OH groups of ser or thr) that
exists as calcium caseinate
- present as micelles in milk
- Serves as a storage protein in milk
- Isolated from milk by isoelectric
precipitation
- Isoelectric pH 4.6
Casein
alpha-s1
alpha-s2
beta
kappa
Whey proteins
alpha-lactalbumin
beta-lactoglobulin
serum albumin
immunoglobulins
other proteins
Alpha-Lactalbumin
- second major protein in bovine milk
- metalloprotein that can bind to several
metal ions like calcium and zinc
- It can serve as a regulatory protein in
lactose biosynthesis
- isolated from whey by heat denaturation
(in acidic condition)
Myoglobin
- Small, bright red protein common in muscle
cells
- Stores oxygen (used when muscles are hard at
work)
- A hemoprotein containing a heme group at its
center
- Isolated by salt precipitation
Myoglobin
Gluten
- Storage protein responsible for the
elasticity and extensibility of dough
- consists of gliadin and glutenin
- Isolated by difference in solubility in
water
- Isolated gluten free of starch when
(-) to iodine test
Reagent:
Principle:
CuSO4 + NaOH
Principle: formation of
coordination complex of Cu2+ and
four nitrogen atoms (two from
each of the two polypeptide
chains)
(+) Result: purple color of solution
(+) Result:
Ninhydrin (triketohydrindene
hydrate)
oxidative decarboxylation &
deamination followed by
condensation
blue-violet color
yellow for proline
(pyrrolidine ring)
HNO3 , NaOH
Principle:
(+) Result:
Reagent:
Principle:
(+) Result:
(+) Result:
Principle:
complexation (base-catalyzed
condensation of a-naphthol
with the guanido group of Arg)
Reagent:
NaOH, nitroprusside
Reagent:
NaOH, (CH3COO)2Pb
Principle:
(+) Result:
NaOH
Principle:
Basic hydrolysis
(+) Result:
Principle:
(+) Result:
Red color
Biuret
Ninhydrin
Xanthoproteic
Millons
Hopkins-Cole
Sakaguchi
Nitroprusside
Fohls
Test for amide
Pauly
Intact
Protein
Protein Hydrolysate
acidic
basic
enzymatic
Paper Chromatography
- Used to determine the amino acid
composition of a given protein solution
- Visualized using ninhydrin
- Retention factor, RF
RF = distance travelled by the amino acid,
cm
distance travelled by the solvent, cm
Bradford Assay
-colorimetric method for determining protein
concentration
- involves use of Coomassie Brilliant Blue G-250
(dye), which reacts primarily to basic (especially
arginine) and aromatic amino acids
-measures 10-100 mg protein
-standard used: bovine serum albumin (BSA)
-Bradford reagent:
dye dissolved in ethanol and phosphoric acid
Bradford Assay
Steps in quantifying proteins using Bradford assay:
1. Prepare BSA standards with different concentration
- stock solution added with different amounts of water
- final concentration of standard is computed using
C1V1 = C2V2
2. Read A595 of standards and samples
3. Plot standard curve
Absorbance (y) vs. Concentration (x)
4. Draw the best fit line
5. Determine the concentration of sample from the
standard curve by extrapolation
Bradford Assay
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