EXPERIMENT 2: POSTLAB

QUALITATIVE TESTS FOR PROTEINS

Recall:
• The solubility of proteins in water is dependent on shape.
o Globular proteins are more soluble in water than fibrous.
• Proteins have varied solubilities in water, dilute aqueous solutions of acids and bases, organic solvents such
as alcohol and some are insoluble even in ordinary solvents.
• They are colorless, tasteless and odorless.
• They are amphoteric. (can be both acid (-COOH) or base (-NH2))
• Characteristic color reactions/tests are dependent on the specific amino acid that makes up the protein.
• Proteins may be denatured permanently (irreversible) or temporarily (reversible).

Classification of Proteins: According to Composition (most important classification in proteins)

1. Simple proteins
• yield only amino acids upon hydrolysis
• Albumin
o Soluble in water and neutral salt solution; denatured by heat and saturation with (NH4)2SO4;
ovalbumin in egg white; serum albumin in blood
o albumin was salted out to the solution; ammonium sulfate has more affinity in water and will remove
albumin in water
• Globulins
o Insoluble in water but sol in diluted neutral salt solution; denatured by Heat, ½ saturation with
(NH4)2SO4 and by saturation with NaCl; Serum globulin in blood, ovoglobulin in egg white, myosin in
muscle
• Glutenins
o Insoluble in water and diluted neutral salt solution but soluble in dilute acid and alkali; denatured by
heat; Glutenin in wheat and oryzenin in rice
• Prolamines
o Insoluble in water, sol in 60-80% EtOH; no suggestion how to denature it; Gliadin in wheat, zein in
corn, bordein in barley and secalin in rye.
• Albuminoids/scleroproteins
o Insoluble in water, dilute neutral salt solution, acid, alkali and 60-80% EtOH (not soluble at all even
in natural or normal solvents); Hydrolyzed (special process) by long boiling with conc acid solutions;
Elastin in tendons, keratin in hair and horny tissue, fibroin in silk
• Histones
o Soluble in water and dilute acid, insoluble in NH3; no suggestion how to denature; Protein in the
nucleus
• Protamines
o Soluble in water, dilute acid and NH3; no suggestion how to denature; Sturin and salmin in sperm of
fish

2. Conjugated Proteins
• simple proteins + non-protein component
• Chromoproteins
o linked with colored compound.
o E.g. Hemoglobin in blood (heme gives a characteristic color of the blood which is red; hemoglobin
is a metalloprotein)
 • Glycoproteins
o linked with a carbohydrate.
o e.g. mucin in saliva
• Phosphoproteins
o linked with phosphoric acid.
o e.g. casein in milk and vitellin in egg yolk
• Nucleoproteins
o linked with nucleic acids.
o e.g. nuclein found in nucleus of cells
• Lipoproteins
o linked with lipids.
• Metalloproteins
o linked with metals

Classification of Proteins: According to Shape

1. Globular proteins
• polypeptide chain/s folded into spherical or globular shape
• soluble in aq. System
• e.g. enzymes
2. Fibrous proteins
• polypeptide chains arranged in long strands or sheets
• water insoluble, resist digestion (regard to denaturation of albuminoids/scleroproteins)
• e.g, keratin, collagen, fibroin

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

Denaturing agents
Proteins can be denatured by:
a. Strong acids and bases
b. Organic solvents
c. Detergents (surfactants)
d. Reducing agents
e. Salts
• Salting-in
• Salting-out
f. Heavy metals
g. Temperature
EXPERIMENT PROPER
Isolation of Proteins

Milk

• said to be a complete food because it contains all the nutrients we need to have
• Sheep milk has the highest content of fats
• Human and horse milk have the highest content of water among the others
• o/w type emulsion where the fat is finely dispersed.
oAn emulsion
oThere will be a separation of milk; upper portion: fat globulin (cream portion/cream line); lower portion:
plasma phase (skim milk)
• Contains all the common amino acids
• Contain 3 proteins; casein, lactalbumin and lactoglobulin.
• Has pH of 6.6

Isolation of casein
• Casein is a conjugated protein (phosphoprotein).
• PO4—3 interacts (ionic) with Ca+2 leading to polymerization of casein: [casein – P – Ca+2 – P – casein]
o Casein is interact with the elemental calcium (ionically) and phosphorus
• from the latin word of CHEESE
• Principle involved in isolation
o Isoelectric precipitation
▪ adjusting the pH of the solution to the IpH of the protein, decreasing its solubility
• IpH of casein = 4.6, acidic protein (reach the IpH (at its zero charge) so that it can be precipitated out of
the solution)
o Going lower and higher with 0 charge will lead to dissolving the of the proteins
• Calcium caseinate form insoluble (can be bought in the form of tablets as supplements), non-crystalline
white curd when acidified (smell sour since acetic acid was used)

Isolation of albumin
• Albumin is the most abundant protein in the body.
• medically used as albumin volume replacement
• Albumin is a globular protein.
• Has ipH of 7.4
• Principle involved in isolation:
o Denaturation with heat
▪ heat precipitates albumin (at 75 C)
▪ Maintain at 40C so that albumin will not precipitate
Isolation of gluten
• It is a mixture of two proteins: glutenin & gliadin.
• Gluten is a strong and flexible molecule. In bread-making: it traps the CO2 produced by the reaction of flour
& yeast and gives flour its characteristic chewiness.
• Principle involved in isolation
o Solubility Difference
▪ starch is partially soluble in H2O while gluten is insoluble in H2O.
• I2 solution is used to test the complete removal of starch.
• It is a yellowish-white solid, tough, elastic, and sticky

Isolation of myoglobin
• It is a globular protein that is for O2 storage and transport in vertebrates, primarily in heart and skeletal
muscles. (indicator of heart and skeletal muscles diseases)
• It is a monomer made up of 153 AA residues.
• Principle involved in isolation:
o Salting out
▪ addition of (NH4)2SO4 increases the ionic strength of water (increases its polarity) resulting to
decrease solubility of proteins
• Isolated myoglobin is a white precipitate.

Hydrolysis of Proteins: Chemical Hydrolysis

Hydrolysis
• Breaking of peptide bonds; color reaction will be dependent on the amino acids
• Catalysts (eg. acid, base enzyme) are needed to facilitate the hydrolysis of peptide bonds
• This is done to release individual amino acids that will be the basis of color reactions.

Acidic Hydrolysis
• causes complete hydrolysis of proteins.
• The isolated protein was treated with excess 6M HCl and was autoclaved at 15psi for 5hrs.
• Advantages
o Little or no racemization
▪ Racemization
- conversion from L isomers to D isomers
• Disadvantages
o W (tryptophan) is destroyed and is converted to humin (black precipitate);
o partial destruction of S (serine), T (threonine), C (cysteine) ; and
o hydrolysis of N (asparagine) to D (aspartic acid) & Q (glutamine) to E (glutamic acid)

Alkaline/Basic Hydrolysis
• causes complete hydrolysis of proteins.
• Isolated protein was treated with conc. NaOH soln and was autoclaved at 15psi for 5hrs.
• Advantages
o W (tryptophan) is stable
• Disadvantages
o Racemization of all amino acids;
o R (arginine) decomposes to ornithine and urea;
o Partial destruction of C (cysteine), C-C (cystine), S (serine), T (threonine)
Hydrolysis of Proteins: Enzymatic Hydrolysis
Enzymatic Hydrolysis
• proteolytic enzymes cause partial or selective hydrolysis of polypeptide to yield a mixture of peptide
fragments (longer than what we get in acid and alkaline hydrolysis).
• Proteases/Peptidases
o are enzymes that hydrolyze peptide bonds at specific sites
• Advantages
o Amino acids are not affected;
o it requires certain temperature & pH conditions for optimum activity of enzymes.
o Hydrolysis not complete.
Classification of Amino Acid: Polarity
Amino Acids

• Proline has the unique structure

among the non polar alkyl
groups, composing of a closed
ring structure with amine. Take
note that proline has NO
ALPHA AMINO GROUP.
 Classification of Amino Acid: Structure
(M & C) Aromatic (W, Y, P)
Qualitative Color Reactions
● It is very important to take note first the color of the reagent used, whether it is clear, has suspended
particles, because you are expecting a significant change in their colors when conducting Qualitative Color
Reactions
Biuret Test
● General Test for INTACT PROTEINS and PROTEIN HYDROLYSATES (Test for PEPTIDE LINKAGES;
AT LEAST UP TO TRIPEPTIDE)
○ Biuret test is a good test to know whether you completely hydrolyze or not your proteins.
○ If the protein has been completely hydrolyze, it will result into a negative result
○ Reason: Biuret test is sensitive to detect the peptide linkages
○ Sensitivity: At least up to TRIPEPTIDE (2 Peptide linkages and MORE will give you a positive result)
(AA-AA-AA)
○ Dipeptide linkage will give you a negative result (AA-AA)
● Reagent: 2.5M NaOH and 0.1M CuSO4 (Blue in color)
● Principle: Formation of coordination complex of copper and four nitrogen atoms

● Positive Result: Pink to Violet color

○ Blue indicating the presence of CuSO4 or it can show you the complete
hydrolyzation of the proteins, while the violet color represents the presence
of a tripeptide linkages or more.
Ninhydrin Test
● General test for amino acids with a-amino group
● Prolines have no alpha amino group, therefore it will not give a positive result
● Reagent:
○ 1,2,3 - indanetrione monohydrate or triketohydrindene hydrate and ethanol
○ The reagent is a yellow solution
● Principle:
○ Oxidative decarboxylation and deamination followed by condensation
○ The amino acid is first being decarboxylated and then being deaminated and then having the Nitrogen
bridging the two ninhydrin molecules

● Positive Result:
○ Blue to blue-violet
Xanthoproteic Test
● General test for aromatic amino acids
● Xantho means yellow and proteic refers to protein
● This is also why the skin turns yellow when in contact with nitric acid because the skin contains proteins,
aromatic amino acids and they are being nitrated by nitric acid
● Reagent:
○ Conc. Nitric acid and NaOH
● Principle:
○ Nitration of the aromatic rings via SEAr (Aromatic electrophilic substitution)
○ Aromatic electrophilic substitution is where one H from the benzene ring is being replaced by the nitrate

● Positive Result:
○ Yellow Solution/ppt
○ Lemon yellow upon heating
○ Orange with excess NaOH (burnt orange color)
Millon’s Test
● Detects phenolic ring containing amino acid
● Specific test for tyrosine

● Reagent:
○ Millon’s rgt. (Hg(NO3)2 and Hg(NO2)2)
● Principle:
○ Complexation (mercuration & nitration)
● Positive Result:
○ Old rose/reddish brown/flesh colored precipitate
Hopkins-Cole Test
● Detects indole group containing amino acid
● Reagent:
○ Glyoxylic acid (Mg Powder, oxalic acid and Acetic
acid) and Sulfuric Acid
● Principle:
○ Reduction of oxalic acid to glyoxylic acid and acid-catalyzed condensation of two tryptophans with
glyoxylic acid
○ Formaldehyde represents the glyoxylic acid below and the two tryptophans are being condensed acid
catalyzed, upon adding sulfuric acid

● Positive Result:
○ Pink to violet
interface
Sakaguchi Test
● Detects un- or monosubstituted guanidine
● Detects the guanidino
● Reagent:
○ A-napthol, NaOBr, NaOH, Urea (to stabilize the color and
destroy excess OBr- ions)
● Principle:
○ complexation; based-catalyzed condensation of a-napthol with guanido group of Arg
● Positive Result:
○ Red to red orange color

Fohl’s Test
● Detects sulfur-containing amino acids
● Only two sulfur-containing amino acids
● Sulfur in the amino acid is being removed
● Reagent:
○ Lead Acetate and NaOH
● Principle:
○ Degradation and substitution reaction to form PbS
● Positive Result:
○ Brown to black precipitate (Lead sulfide)
○ Methionine does not give the result immediately unless it is heated with a mineral acid to release the
sulfur

Nitroprusside Test
● Detects cysteine
● Reagent:
○ Na2Fe(CN)5NO in dil. NH3 and
NaOH
● Principle:
○ Complexation reaction

● Positive Result:
○ Red coloration
○ Most usually the thiocyanate is responsible for the red color in nitroprusside test

Test for Amide

● Detects primary, secondary and tertiary amines and nitriles
● Reagent:
○ NaOH
● Principle:
○ Basic Hydrolysis
○ Upon hydrolysis of the amides, the amines and nitriles are being released in the form of
ammonia
● Positive Result:
○ Red-blue litmus paper
● When you are heating a tube and you have a red litmus paper and you put NaOh , the fumes will turn the
red litmus paper to blue indication that a basic material has been released from the heated solution and
there is only one material that can do that which is your ammonia
Pauly’s Test (Ehrlich’s Diazo)
• Detects TYR and HIS
• Reagent
o Diazo Reagent (1% SULFANILIC ACID WITH 5% NaNO2 ) AND 10% Na2CO3
• Principle
o formation of azo dye detected by the red coloration
• Positive result:
o Red coloration
▪ His is red to orange;
▪ Tyr is light orange
Chemical Intact
Detected Group Protein Hydrolysates
Test Protein

H+ OH- Protease

-/+
- if complete hydrolysis
Biuret Peptide linkages + + If partial hydrolysis upon
-/+ +
using the acid

All AA having free –

Ninhydrin
NH2 except proline + + + +

Xanthoproteic Aromatic rings (WYF) + + + +

-
Acids are able to destroy
Hopkins-Cole Indole group (W) + tryptophan that is why there + +
is a negative result when
testing the acid hydrolysate

Millon’s Phenolic group (Y) + + + +

-
Arginine is being
Sakaguchi Guanidine group (R) + + destroyed when using
+
alkalize to hydrolyze

Fohl’s Test S group (M*C) + +/- +/- +

Nitroprusside SH group (C) + + - +

Amide test Amide group + + + +

+/-
The acid and alkali used to
Pauly’s Test Y and H + hydrolyze the proteins + +
partially destroys the
cysteine and methionine

Thin Layer Chromatography

TLC
● This technique is used to separate and identify components/solutes in a mixture.
● Principle Involved: difference in the affinity of the solute on the mobile and stationary phase.
● TLC classified under normal phase and partition chromatography.
○ Mobile phase
▪ Butyl alc:HAc:H2O (4:1:5) BAW, UPPER LAYER
 ○ Stationary phase
▪ TLC COATED SILICA GEL (polar) - the thin film is coated with silica gel (polar)
▪ Amino acids may be classified as polar and nonpolar and that will be the basis for their migration or
their result in the TLC.

Steps in TLC
● Sample/Standard Application
○ small spots of std/sample are applied to avoid overlapping and tailing during development
● Development
○ equilibration (saturation of chamber with mobile phase) to hasten development;
○ equilibrate first the chamber that is to saturate the chamber with the mobile solvent so the development
can be faster
● Visualization
○ chemical visualizing agent:
▪ ninhydrin spray for amino acids and proteins (Proteins are colorless so generally the AAs standard
we are using for TLC are colorless.
▪ There is now way for you to see them without using the visualizing agent. )
● Evaluation
○ comparing Rf values of sample and standards
● Documentation
○ chromatogram (finished TLC with spots)

Rf = distance travelled by the sample/distance traveled by the solvent

*Start with the TLC plate then draw lines: origin - here is where you will put the samples and the solvent front -
the development or process is finished if the solvent has reached the solvent front. Remove from the chamber
and allow it to dry and then go for visualization. The basis of TLC is polarity.

*You will only see color on the spots after you spray with ninhydrin and you have dried it over the hot plate a
little.
*Silica gel that is contained at the TLC plate is polar. The tendency is that the polar amino acids could not rise
in the TLC plate
*Refer to the right picture: Arginine, aspartic acid, cysteine have more affinity with the silica gel meaning they
are more polar compared to those who have rise like Tryptophan and Tyrosine (nonpolar)
*Orange: nonpolar amino acids
*Lower pic: Stailing (?) - not a clean spot; shadow sa ilalim
*The Rf values of the polar amino acids are lesser than the Rf values of those that are nonpolar which means
that the solvent used have more nonpolar character so they are able to carry the nonpolar amino acids with
them.

*Hydrolysates - meaning pwedeng hindi totally separated yung amino acids that are in the protein; fragments
ang nakuha that is why there are several separations.
Subject it to UV light to visualize better and to see if there are bonds and fragments.

Experiment Proper: Quantitative Analysis

Quantitative Analysis Of Proteins
● Kjeldahl’s Procedure
● Lowry’s Method
● UV Spectrophotometry Method
● Biuret Method (540 nm)
● Bradford method (595 nm)
● Standard used:
○ Bovine Serum Albumin
○ A standard calibration curve of BSA will be prepared to determine the amount of proteins present in the
sample.
Kjeldahl’s Procedure
● The protein sample is digested by boiling (360°C) with concentrated sulfuric acid in presence of
copper sulfate and sodium sulfate as catalysts.
● The nitrogen present in the protein is reduced to ammonia.
● The liberated ammonia can be calculated.
● Then the quantity of nitrogen originally present in the protein is assessed.
● How are they assessed = Since proteins, on an average contain 16% nitrogen, the weight of nitrogen ×
100/16, or nitrogen × 6.25 will give the value of proteins present in the original sample.
● Remember, you are measuring ammonia, you are producing ammonia and the ammonia becomes the basis
for the protein contents. Multiplying that by this factor, knowing that proteins, on average, would be
containing 16% nitrogen, since all amino acid has nitrogen.
● Advantage
○ This is the most accurate and precise method of quantitative determination.
○ It is generally used for standardizing a particular protein;
○ that protein is then used for calibrating other proteins employing other easier methods.
○ Before you do other tests, or use a certain standard, try it first on Kjeldahl's to establish its amounts or
quantity.
● Disadvantage
○ It takes many days to get the result, and is unsuitable for routine clinical work.

Lowry’s Method
● This is based on the reduction of Folin-Ciocalteau phenol reagent (phosphomolybdic acid and
phosphotungstic acid) by the tyrosine and tryptophan residues of protein.
● A blue color is developed, which is compared with that produced by a known standard.
● Advantage
○ This method is very sensitive and protein content in microgram range can be measured.

UV Spectrophotometry Method
● Proteins will absorb ultraviolet light at 280 nm. This is due to the tyrosine and tryptophan residues in the
protein.
● Quantitation is done by comparing the absorbance of the test solution with a known standard.
● Advantage
○ The method is accurate, simple and highly sensitive up to microgram quantities.

Biuret Method
● Biuret Test
○ qualitative analysis
● Biuret assay method
○ quantitative analysis
○ uses same principle with Biuret test
▪ uses same reagent - Cupric ions chelate
● Cupric ions chelate (complex formation) with peptide bonds of proteins in alkaline medium produce a pink
or violet color.
● The intensity of the color is proportional to the number of peptide bonds.
● More intense the violet color, the more impact the peptide bonds are
● The color is then compared with a standard protein solution treated with the Biuret reagent, and estimated
colorimetrically @540 nm
Biuret method
● Advantage
○ A simple one-step process
○ Most widely used method for plasma protein estimations
○ Easy to use
● Disadvantage
○ Sensitivity is less
○ Unsuitable for estimation of proteins in milligram or microgram quantities
▪ Not ideal if protein content in the sample is too low

Bradford Method
● A simple and one of the most commonly used assay for total protein concentration
● Based on the proportional binding of Coomassie dye to proteins
○ The more proteins present, more dye will bind, more intense the color will be
○ ↑ proteins = ↑ binding of dye = ↑ intensity of color
● It is a colorimetric assay
○ The color intensity of the solution depends on the concentration of protein
● Principle involved
○ In an acidic medium, proteins bind to Coomassie dye (red) due to hydrophobic and electrostatic
interactions
○ Λ max of dye = 465 to 595nm
○ Red solution → Blue solution (stable up to 1 hr)
▪ Red Coomassie dye turns blue once it binds with proteins

Quantitative Protein Analysis

Preparation of Standard BSA

• Two-fold dilution
• Reaction mixture
• Bradford
o no heat needed
o Requires less concentration or dilution of samples
• Biuret
o incubation at 60 °C in water bath for 10 minutes to develop color in sample
o Requires more concentration or dilution of samples
Comparison between Biuret and Bradford Assays
CRITERIA BIURET BRADFORD

Light blue to Pink-violet or violet Red dye to Coomassie blue

Principle
involved
Complex of Cu Binding with Coomassie dye

Wavelength 540 nm 595 nm

For general use, fairly

Accuracy Less accurate than Bradford
accurate

Less sensitive
Sensitive
Sensitivity requires higher amount of sample
Detects up to 1 to 20 ug
Detects up to 0.5 to 2.5 mg

Incompatible with detergents

Incompatible to ammonium salts and
Compatibility reducing agents like Compatible with most salts,
with reagents Dithiothreitol due to the metal (copper) solvents, buffers, reducing
which can be reduced substances and chelating
agents.