EXPERIMENT 2: PROTEINS

ISOLATION OF PROTEINS

CASEIN

Primary structure - Amino acid composition: E (20%), D (96.4%), K (7.4%). R (3.7%), H (2.85%), P, L, V, S
(5.7%), Y, I, F, T, A, M, G, W, cystine (0.3%)

Tertiary structure - Globular protein at any pH except 4.6; dispersible or soluble in water, predominantly
constituent amino acids (or amino acid residues) whose R-groups (polar or ionic) with which
water can physically interact with (“like dissolves like”, hydrogen bond, ion-dipole interaction).

Isolation - Isoelectric precipitation

Ca​++​ caseinate + CH​3​COOH

(CH​3​COO)​2​Ca + Casein

● pH = pI: protein is zwitterionic and least soluble in aqueous medium.

● pH > pI: protein is soluble and has a net negative charge.
● pH < pI: protein is soluble and has a net positive charge.

Organoleptic analysis - White amorphous solid, odorless and tasteless; soluble in water at any pH except 4.6.

Function - Storage protein for nutrients, calcium, phosphorous, and supplies essential amino acids.

- Phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, and

histidine.

LACTALBUMIN

Source - Found in mammalian milk; second most abundant protein in milk.

Primary structure - Amino acid composition: A, R, N, C, Q, E, G, H, I, L, M, P, S, W, T, Y, and V

Tertiary structure - Globular protein; dispersible or soluble in water, whose R-groups (polar or ionic) with which
water can physically interact with (“like dissolves like”hydrogen bond).

Isolation - Heat coagulation (difference in solubility)

Heating at 75​o​C aqueous solution denatures the protein by breaking the hydrogen bonds
in the secondary and tertiary structure exposing the non-polar R-groups of constituent
amino acids making the protein less soluble in the aqueous medium.

Organoleptic analysis - White amorphous solid, soluble in water.

Function - Transport protein for fatty acids and bilirubin, storage protein for essential amino acids.

GLUTEN

Source - 80% in wheat flour.

Structure - Simple protein, consisting of two major protein fractions: glutenin and gliadin, rich in amino
acid constituents with non-polar or hydrophobic R-groups.

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 Primary & tertiary - Both have generally similar amino acid composition but have individual differences.
structures ● Glutenin: with higher proportions of K, G, W, R, Y, T, D, N, S, A; multimeric
aggregates stabilized by intermolecular disulfide bonds, hydrophobic interactions;
has quaternary structure fibrous protein, insoluble in water.
● Gliadin: richer in P, E, Q, F, and I, amide and cystine (monomeric, slightly soluble in
water).
- There is no definite differences for M, V, L, and H.

Isolation - Washing with water (difference in solubility).

The wheat flour dough is washed until starch is removed. Washings are analyzed for
starch by treating the washings with I​2​ reagent (aq). Starch reacts with iodine forming a
soluble violet-black or blue-black complex of starch iodine or iodostarch. The insoluble
material left is gluten. When the flour is mixed with water and kneaded, gluten absorbs
water and partially infolds. Hydrophobic and disulfide interactions facilitate the formation
of a threadlike polymer which may be stabilized further by hydrogen bonding.

Organoleptic analysis - Visco-elastic, white amorphous solid, insoluble in water; has thermoplastic property.

Function - For support, structural protein, gluten is responsible for the strength and elasticity of the
dough. It facilitates leavening of the dough, allowing the dough to rise when baked.

MYOGLOBIN

Source - Muscle tissue - red meat.

Structure - Conjugated protein: chromoprotein; prosthetic group heme pigment = Fe (II) + protoporphyrin
IX; responsible for the red color of the sample.

Primary, secondary, and
tertiary structures
- The protein consists of a single polypeptide chain 153 amino acid residues; has a compact
structure, with the interior atoms very close to each other. It has a B ​α-helical regions
stabilized by H-bonding in the polypeptide chain and no pleated sheet. In the helical regions,
75% of the residues are found. Also, the R-groups are also involved in H-bonding.
● Polar amino acid constituents: exterior of the helix.
● Non-polar amino acid constituents: interior of the protein almost exclusively.
● Polar His residues: found in the interior and are involved in the interaction with the
heme group and bound oxygen and this play an important role in the function of the
protein.
● Globular protein: dispersible or soluble in water; physically interacts with water via
R-group of its constituent amino acids (“like dissolves like”, hydrogen bond, and
ion-dipole interaction).

Isolation - The aqueous solution of the protein is buffered at pH 7.5 so that the protein is isolated in
form.
More than 70% (NH​4​)​2​SO​4 ​(aq) is needed to precipitate out the protein and separate
other proteins as ammonium sulfate crystals is added and dissolved to saturate the
aqueous solution. Other proteins are precipitated out initially on the addition of 70%
(NH​4​)​2​SO​4 ​(aq) and are separated by centrifugation (13,000 g). The proteins come out as
pellet. Myoglobin is extracted in the supernatant liquid (centrifuge). Myoglobin is
salted-out and precipitated by adding more (NH​4​)​2​SO​4 ​to saturate the centrifuge (aq.
solution of the protein). The inorganic salt takes out water from myoglobin and
desolvates the protein by ion-dipole interaction.

Organoleptic analysis - Reddish amorphous solid; dispersible or soluble in water.

Function - Storage protein for oxygen in muscle tissues.

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 AMINO ACIDS AND COLOR REACTANTS

1. BIURET TEST

Description - Biuret is an organic compound produced when urea is heated above its melting point. It
undergoes condensation reaction producing NH​3​ gas (ammonia).

Reagent - Cu​2+​, NaOH (aq): blue solution.

Reaction - Formation of metal ion coordination complex with peptide bond.

Cu​2+​ ion has four empty atomic orbitals in the valence shell. The orbital can
accommodate an electron pair, i.e. the lone pair on the nitrogen atom or a peptide
bond. Ammonium and magnesium salts interfere with this test.

Positive result - Violet coloration.

Limitation - Positive only with peptide compound containing more than 1 peptide bond (e.g. Enzymatic
hydrolysate).

Application - For quantitative determination of proteins.

Can be used for small amount of protein samples in a spectrophotometric assay using
standard or reference protein. As the number of peptide bond increases, more protein
is present in the sample. The absorbance of more product of the reaction is read at
540 nm. The assay is of low sensitivity and requires at least 1 mg of the protein
sample.

2. NINHYDRIN TEST

Reagent - Oxidized ninhydrin (triketohydrindene hydrate) in 95% EtOH; pale yellow solution. The
reagent acts as a n oxidizing and deaminating agent. The reaction requires two
equivalents of the reagent.

Reaction - Oxidative deamination with a free amino group, -NH​2​.

The amino acid is released as ammonia. This reaction is not specific for amino acids.
It is given by ammonia and many amino compounds in which carbon dioxide is not
liberated during the reaction.

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 Positive result - Blue to blue-violet coloration with ​α-amino acids (primary amino group).
- Yellow coloration with cyclic amino acids proline and hydroxyproline.
- Characteristic brown product with asparagine - carbonyl group.
Note: The color deepens as the number of amino groups increases.

Application - For quantitative determination of proteins.

Can be used for small amount of protein samples in a spectrophotometric assay using
standard reference protein. As the number of amino acid increases, more protein is
present in the sample.

3. XANTHOPROTEIC TEST

Reagent - Concentrated HNO​3​ followed by the addition of concentrated NaOH (both reagents are
colorless).

Reaction - Nitration of benzene or phenyl group via electrophilic substitution (S​E​) mechanism.

Positive result - Yellow coloration on heating with HNO​3​ forming nitro derivative; orange coloration when
made alkaline with the base forming sodium salt of these derivatives.

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 - The test is positive with tyrosine and tryptophan. Phenylalanine is not as readily nitrated
and does not or weakly responds to this test as it is ordinarily performed.

Application - For qualitative detection of amino acids with a phenyl group or benzene ring.

4. MILLON’S TEST (MILLON’S-NASSE TEST)

Reagent - HgSO​4​, Hg in HNO​2​ colorless.

Reaction - Formation of a colored complex which is probably the salt of the phenolic compound.
Considerable quantities of inorganic salts like chlorides or alkali interfere by
precipitating the mercury salt of the reagent. This is overcome by adding NaNO​2​ or
more of the reagent.

Positive result - Old rose or pinkish or flesh coloration.

Application - For qualitative detection of phenolic group or phenolic compound which is unsubstituted at
the 3-, 5-positions such as tyrosine, phenol, and thymol.

5. HOPKIN’S-COLE TEST

Reagent - Mg​0​ and cold oxalic acid to produce glyoxylic acid, conc. H​2​SO​4​ is added slowly along the
side of the test tube.

Reaction - The indole group in tryptophan condenses with formaldehyde derived from glyoxylic acid in
the presence of H​2​SO​4​.

Positive result - Formation of violet coloration or violet ring at the interphase of two layers as the acid is
added slowly.

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 Application - For quantitative detection of indole group in amino acids.

6. SAKAGUCHI TEST

Reagents - Added in the following order:

1. 10% NaOH (aq): colorless, neutralizes acid groups like the protonated amino
group in the guanido group.
2. 0.02% ​α-naphthol in 95% EtOH: Sakaguchi reagent, colorless; reacts with
guanido group.
3. 2% NaOBr (aq): yellow; acts as oxidizing agent.
4. Urea: white colorless crystals; stabilizes the amino acid, by preventing its
hydrolysis.

Reaction - Substances with guanido or guanidino group react with ​α-naphthol and an oxidizing agent.

Positive result - Yellow coloration on heating with HNO​3​ forming a nitro derivative.
- Orange coloration when made with base forming sodium salt of these derivatives.

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 Application - For qualitative detection of amino acids with guanido group. Arginine whether free or
combined reacts with the test.

7. FOHL’S TEST (LEAD ACETATE TEST, REDUCED SULFUR TEST)

Reagent - 6M NaOH, lead acetate {Pb(CH​3​COO)​2​} crystals.

Reaction - Fusion with NaOH followed by ionic reaction with lead acetate.
Covalently bonded sulfur is converted by fusion with a strong alkali (NaOH) to sodium
sulfide which ionizes in an aqueous medium to produce a sulfide ion (S​-2​). The sulfide
reacts with lead acetate to produce lead sulfide.

Positive result - Formation of brown-black precipitate.

Application - For qualitative detection of sulfur in cysteine and cystine. The sulfur in methionine is not
readily removed by the alkali and is not affected by the reaction.

8. PAULY’S TEST (DIAZO REACTION)

Reagent - 5% NaNO​2​, 1% sulfanilic acid, 10% Na​2​CO​3​.

Reaction - Diazotization.
Covalently bonded sulfur is converted by fusion with a strong alkali (NaOH) to sodium
sulfide which ionizes in a n aqueous medium to produce a sulfide ion (S​-2​). The sulfide
reacts with lead acetate to produce lead sulfide.

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 Positive result - Formation of red or purple coloration.

Application - Tyrosine and histidine forms red or purple condensation product.

This coupled with amines phenols and imidazoles to form highly colored compounds,
the monoazo and bi-azo products. The latter has been shown to predominate at
higher pH.

9. NITROPRUSSIDE TEST (DIAZO REACTION)

Reagent - Na​2​FeNO(CN)​5​ (aq) sodium nitroprusside (green).

Reaction - Acid-base reaction.

Positive result - Deep red or purple color.

Application - Detects cysteine cia its -SH (thiol, sulfhydryl, or mercapto group).

10. TEST FOR AMIDES

Reagent - 20% NaOH, moistened Litmus paper (red).

Reaction - Alkaline hydrolysis via S​N​acyl​.

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 Positive result - Moistened red litmus paper turns blue, basic reaction.

Application - Detects asparagine and glutamine.

HYDROLYSIS OF PROTEINS

ACID HYDROLYSIS

Catalysed by - Strong acid: 6M HCl.

Process - Reaction mixture was autoclaved at 120​o​C, 15 psi, for 5 hours.

- Neutralized and checked with red and blue Litmus paper.

Reaction mechanism - Takes place via S​N​acyl ​mechanism, hydrolysis of amide.

Effects 1. Complete hydrolysis producing L-amino acids.

2. Little or no racemization.
3. Not all amino acids are recovered quantitatively; Trp is destroyed, some loss of Ser and
Thr.
4. The amide group of Asn and Gln undergoes complete hydrolysis yielding Asp and Glu plus
free ammonia.

Color - Color after hydrolysis is brown-black or black amorphous solid called HUMIN or MELANIN.
This is due to condensation of Trp with an unknown aldehyde which may be a

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 carbohydrate.

ALKALINE HYDROLYSIS

Catalyzed by - Strong base: 6M NaOH.

Process - Reaction mixture was autoclaved at 120​o​C, 15 psi, for 5 hours.

- Neutralized and checked with red and blue Litmus paper.

Reaction mechanism - Takes place via S​N​acyl ​mechanism, hydrolysis of amide.

Effects 1. Complete hydrolysis producing L-amino acids.

2. Racemization occurs thus not optically active.
3. Not all amino acids are recovered quantitatively; Cys, cystine, Ser, and Thr. Trp is stable.
4. The amide group of Asn and Gln undergoes complete hydrolysis yielding Asp and Glu plus
free ammonia.
5. Arg is hydrolyzed to ornithine and urea.

ENZYMATIC HYDROLYSIS

Catalyzed by - Hydrolases: proteinase, protease, or proteolytic enzymes.

Process - For chymotrypsin and trypsin (both endopeptidase):

Secondary buffer is added to maintain the pH to 7.5. The reaction mixture is incubated
at 37​o​C.

Reaction mechanisms ● Chymotrypsin: cuts the C-side of the following amino acids: Trp, Tyr, Phe.
● Trypsin: cuts the C-side of the following amino acids: Arg and Lys, provided that (N-ter to
C-ter) is not proline (because it will not fit the active site).
● Papain/Papase: a Cys endopeptidase; independent of pH and heat resistant (60-70​o​C);
catalyzes hydrolysis of amino acids with bulky hydrophobic or aromatic group at the
carboxyl side provide that the next amino acid is not valine.

Effects 1. Incomplete hydrolysis, selective hydrolysis.

2. Enzymes are reaction-specific and substrate-specific.

ANALYSIS OF RESULTS ON HYDROLYSIS OF PROTEINS

1. The chemical test may be negative for sample (intact) even if the functional group for the positive result is present. The
group may not be exposed on the surface of protein.
2. If the chemical test is positive for the intact protein, the result would be more intensely positive after hydrolysis.
3. Biuret test is positive for the sample before hydrolysis but definitely negative after basic and acid hydrolysis. It may be
positive after enzymatic hydrolysis.
4. Ninhydrin test is positive for the sample before and after hydrolysis. The result after hydrolysis is more intense.
5. Hopkin’s-Cole test is definitely negative after acid hydrolysis even if the intact protein is tested positive before hydrolysis.
6. Sakaguchi test is definitely negative after basic hydrolysis even if the intact protein tested positive before hydrolysis.
7. The enzymatic hydrolysate contains a mixture of L-amino acids and smaller peptides. All the amino acids are recovered
quantitatively. In general, hydrolysis is incomplete, not all peptide bonds are broken.

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 CHROMATOGRAPHIC ANALYSIS OF PROTEIN HYDROLYSATES

Purpose - Used for separation and qualitative analysis; identify the amino acids in the hydrolysate by
comparison to Rf values.

General principle - Separation of components in a mixture is based on the difference in affinity for or
distribution between two phases: stationary and mobile phases.

THIN LAYER CHROMATOGRAPHY

Stationary phase - Silica gel coated on Al metal sheet; has silanol groups (Si-OH).

Mobile phase - N-butanol : acetic acid : water (4:1:5).

Use upper layer.

Principles - Normal phase chromatography where the stationary phase is more polar than the mobile
phase.
- Non-polar or less polar components will migrate at a faster rate and at a higher distance
from the origin through the stationary phase than the more polar components.
- The non-polar components are more soluble in the mobile phase than the more polar
components.
- The less polar amino acids migrate faster from the origin, thus, have higher Rf values.

Factors contributing to the 1. Number of carbon atoms: less carbon = more polar.
polarity of amino acids 2. Polar or ionic R-groups: ionic > polar.

Rf values - Defined as the ratio ​of the distance moved by the solute and the distance moved by the
solvent.

Rate flow, retardation - The higher the Rf value, the greater the affinity to the mobile phase.
factor, retention factor, or - The lower the Rf value, the greater the affinity to the stationary phase.
relative to the front

TLC chromatogram

- Colvent system: n-butanol : acetic acid : H​2​O (4:1:5).

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 Rf values of amino acids

BRADFORD ASSAY

Spectrophotometry - Based on the amount of light absorbed by the compound.

- The amount of light absorbed (absorbance) by the compound is inversely proportional to
the light transmitted (transmittance).

A = -log T or A = log 1/T

Beer-Lambert’s Law - States that the amount of radiation absorbed is directly proportional to the concentration of
the absorbing species and to the length of the light path.

A = abc
A = optical density
a = molar absorptivity (L . mol​-1​cm​-1​)
b = distance traveled by the light (cm)
c = concentration of the absorbing species (mol . L​-1​)

Wavelength ranges for

colors

For quantitative analysis ● Single Standard Method

As = absorbance of standard solution

Ac = absorbance of unknown solution
Cs = concentration of standard solution
Cu = concentration of unknown solution

● Multiple Standard Method (Calibration Curve)

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 Dye used - Coomassie Brilliant Blue G2-250 (CBB G-250), a protein binding dye.

Process and Principles
1. The assay is based on the immediate absorbance shift from 465 nm to 595 nm upon
binding of the dye to protein in acidic medium.
2. CBB G-250 dye interacts chiefly with His, Lys, Tyr, Trp, Phe. This dye binds
non-specifically to all proteins.
3. The dye donates a proton (H+) to ionizable R-group on the protein like - NH​2​, making the
group positively charged. This allows the blue form of the dye to ionically interact with the
protonated amino groups of the protein resulting to absorbance shift from 465 to 595 nm
(red to blue).
4. It is:
Highly sensitive,
Highly reproducible.
Very fast.
5. It detects 1-200 ​μg protein.
6. It uses Coomassie Brilliant Blue G2-250, a protein binding dye.
7. It works well even in the presence of urea or guanidine hydrochloride, but not in the
presence of detergent.

TWO METHODS

1. Graphical Method 1. Plot the concentration (x-axis) versus absorbance (y-axis).

2. Draw a straight line passing through an area where most of the points are concentrated in
the X-Y plot.
3. One may start at zero intercept to produce a straight line. Use a ruler.
4. Interpolate as illustrated.

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 2. Linear Regression 1. Plot the concentration (x-axis) versus absorbance (y-axis).
Analysis 2. Use the linear equation, y = mx + b, to get the value of x, which represents the
concentration of the unknown.

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