PHA6112 - Lab - Proteins

UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY BATCH 2025 PHA6112

LAB:PHARMACEUTICAL BIOCHEMISTRY
PROFESSOR IRISH MHEL C. MITRA, MSc
PROFESSOR RACQUEL C. CRUZ, MSPh
air and in the soil.
o Animals cannot synthesize proteins
Intended Learning Outcome from such materials.
I. At the end of this chapter the o Animals obtain proteins from plants or
student must be able to: from animals which obtain the same
from plants.
II. Apply the appropriate
techniques for the isolation,
Proteins: Classification
purification and characterization
of proteins
According to Function
III. Predict the primary structure of a
2. According to Composition
protein based on enzymatic
3. According to Shape
reactions and chemical
4. According to Solubility
hydrolysis.
According to Composition
Outline Simple proteins – yield only amino acids upon
● Overview of proteins hydrolysis
● Protein isolation e.g. albumins and globulins
○ Casein Conjugated proteins – yield simple proteins and
○ Albumin a nonprotein material (prosthetic group) e.g.
○ Gluten nucleoproteins, glycoprotein, lipoprotein,
○ Myoglobin phosphoprotein, hemoprotein, metalloproteins
● Analysis of proteins
○ Qualitative According to Shape
○ Quantitative
Globular proteins – polypeptide chain/s
folded into spherical or globular shape; water
soluble
Proteins
e.g. enzymes
Proteins Fibrous proteins – polypeptide chain/s
arranged in long strands or sheets; water
o Large biomolecules that primarily insoluble
contain C, H, O, and N. e.g. keratin, collagen
o Some contain nonmetals S, P and I.
Others contain metals According to Solubility
such as Fe, Cu, or Mn.
o Made up of simple molecules - amino Albumins – soluble in water and dilute aq
acids. solutions
o Sources: Globulins – soluble in dilute salt solutions but
o Plants synthesize proteins from are insoluble or sparingly soluble in water.
inorganic substances present in the o
ABSALUD,BARROGA, CUISIA, DE PABLO, GATAPIA, MANGANTI, SAN JUAN, VIZ 1 I 2BPH (A.Y.
2022-2023
)

FACULTY OF PHARMACY BATCH 2025
PHA6112 LAB:PHARMACEUTICAL BIOCHEMISTRY
Glutelins – soluble in dilute solutions of acids

and bases; insoluble in neutral solvents.
Prolamins – soluble in 50-90% alcohol,
insoluble in water, neutral solvents or absolute
alcohol
Albuminoids/scleroproteins – insoluble in When a protein is hydrolyzed (by acids, bases
most ordinary solvents or certain
enzymes), it breaks down into smaller units,
Proteins: Physical Properties eventually forming amino acids
Additional notes:
Generally odorless, tasteless, and colorless Few ● Once exposed to acid, NH2
are colored crystalline solids with relatively becomes NH3+.
high MP ● Once exposed to base, COOH
Form colloidal dispersions and do not pass will become COO-..
through
membranes.
Have varied solubility in acid, base, and salt
solutions.
LMW proteins are mostly soluble while HMW
proteins are Give specific applications for different types of
solid tubed media
insoluble.
Additional notes:
● Use of acid = complete
PROTEINS: Chemical Properties
hydrolysis ○ Protein will be
Amphoteric in nature
cut into its
• Contains –COOH group and –NH2 thus, can
monomers.
react
● Use of enzyme = partial hydrolysis ○
chemically as either acid or base
Protein will be cut down into
• This accounts for their ability to act as buffers
peptides.
in the blood

Additional notes: Protein Denaturation is the loss of
● Proteins in crystalline form are not as
three-dimensional structure
sensitive to temperature, so they
sufficient to cause loss of function.
do not denature easily.
Types:
1. Irreversible Denaturation
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)

point where it becomes
insoluble.
- biological function /activity cannot be
● The capacity of alcohol to
regained
precipitate bacterial proteins makes
2. Reversible Denaturation
it function as a disinfectant.
- biological function/activity can be regained
● In the laboratory, ethanol and
isopropyl alcohol are used.
Additional notes:
○ Optimal precipitation using
● Irreversible denaturation is achieved
through heating. alcohol - 20°C.
● Reversible denaturation is achieved
by adjusting isoelectric pH.
○ Protein will regain function Points to Remember
once optimum conditions ● Techniques and principles involved in
are restored. the isolation of proteins.
● Qualitative test and principles in the
identification of amino acids
• Proteins are precipitated by:
● Techniques and principles in acid,
• Heat
alkaline and enzymatic hydrolysis
• Alcohol: precipitates bacterial proteins
● Application of Thin Layer
• Strong acids: HCl, HNO3
Chromatography in the identification
, and H2SO4
of amino acids
• Salts of heavy metals: HgCl2
● Quantitative total protein analysis
, CuSO4
, Pb(CHCOO)2
• Alkaloidal reagents: Trichloroacetic acid (TCA),
picric acid,
phosphotungstic acid
• Others: detergent, reducing agents and salts
ISOLATION OF PROTEINS
● CASEIN
Additional notes: ● ALBUMIN
● Heat causes irreversible ● GLUTEN
denaturation. MYOGLOBIN
○ Since it can cause ● ISOLATION OF CASEIN FROM
intramolecular SKIMMED MILK
rearrangement and can ● PROTEINS
decrease solubility to the ● Casein, lactalbumins, and
lactoglobulins are globular proteins an acid (Acetic acid) to adjust the pH
found in milk. Casein exists in milk as of the sample to 4.6, the isoelectric pH
calcium caseinate. of casein.
● Milk has a pH of 6.6.
● Isolation of casein is carried out using
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)

interacts (ionic) with Ca+2 leading to
polymerization of
casein:
MATERIALS AND EQUIPMENT NEEDED ●
[casein – P – Ca+2 – P – casein]
Powdered, non-fat milk (any brand) ●
10% CH3COOH (any but preferably
cane vinegar) Additional notes:
● Thermometer (electronic thermometer) ● At the isoelectric pH, protein
● pH meter/indicator becomes insoluble, hence the
● Funnel and Filter paper (strainer with formation of precipitate.
cheese cloth or coffee
● filters)
● Beaker (any small glass casserole that
Principle involved in isolation: Isoelectric
is heat resistant)
precipitation –
● Water bath (casserole with water)
adjusting the pH of the solution to the IpH of
the protein,
Additional notes: decreasing its solubility
● Double boiling is the preferred • IpH of casein = 4.6, acidic protein
method of heating in the • Calcium caseinate form insoluble,
home-based experiment since noncrystalline white curd when acidified.
direct heat could cause abrupt
temperature changes. ISOLATION OF ALBUMIN FROM SKIMMED
● Beyond 40°C, albumin will be MILK
co-precipitated. ● Albumins are globular proteins that are
soluble in water and dilute salt
ISOLATION OF CASEIN FROM SKIMMED MILK solutions and denatured and
coagulated by heat
ISOLATION OF ALBUMIN FROM SKIMMED

MILK
MATERIALS AND EQUIPMENT NEEDED ■
Filtrate/supernate from the isolation of
ISOLATION OF CASEIN casein
• Casein is a conjugated protein ■ Thermometer
(phosphoprotein). PO4 —3
■ Beaker (any heat-resistant glass
casserole)
■ Hot plate (pan with water)
Additional notes:
● Presence of fat could affect the
results, hence the use of non-fat
milk.
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)

● 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.
ISOLATION OF ALBUMIN
• Albumin is a globular protein.
• Has ipH of 7.4
• Principle involved in isolation: Denaturation
with heat – heat precipitates albumin
Additional notes: ISOLATION OF GLUTEN

● Presence of starch can be detected ● Principle involved in isolation:
using the iodine solution due to the Solubility Difference– starch is partially
formation of the iodo-starch soluble in H2O while gluten is insoluble
complex. in H2O.
● I2 solution is used to test the complete
ISOLATION OF GLUTEN FROM WHEAT removal of starch.
FLOUR ● It is a yellowish-white solid, tough,
Gluten is isolated from wheat flour by elastic, and sticky
washing the dough with water. This process ISOLATION OF MYOGLOBIN FROM MUSCLE
removes excess starch and insoluble material
remaining is the gluten ● Myoglobin and hemoglobin are vital for
oxygen transport and storage.
MATERIALS AND EQUIPMENT NEEDED ● Myoglobin is present in large
1 cup wheat flour quantities in muscles and responsible
Cheesecloth for the red color of the tissue.
0.01 M Iodine solution (tincture of iodine ● Myoglobin is isolated thru ammonium
or betadine) sulfate
precipitation method (salting out method)
● 70% buffer-diluted (NH4)2SO4
ISOLATION OF MYOGLOBIN FROM MUSCLE solution, pH 7.5
MATERIALS AND EQUIPMENT NEEDED ●
Minced beef/steak + 6 mL of 70% (NH4 )2SO4
● Cheesecloth ● Stir the mixture for 1 minute
● Centrifuge ● Express the dark-red extract in a
● Centrifuge tubes beaker using a cheesecloth
● (NH4)2SO4 crystals
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)

Salting out addition of (NH4
)2SO4
● Centrifuge the filtered solution @4000
increases the ionic strength of water resulting
rpm for 5 minutes. to decrease solubility
● Transfer the 10 ml of the supernatant of proteins
into another empty
• Isolated myoglobin is a white precipitate.
● centrifuge tube
● Add 3 ml of saturated solution of ISOLATION OF CASEIN, ALBUMIN, GLUTEN
ammonium sulfate. Home-based Experiment
● Mix gently and avoid frothing incubate PROTEINS
in an ice bath for 20 mins ● Two members shall perform isolation of
● Centrifuge and decant off the casein and albumin.
supernatant and describe the ● Four (or five) members shall perform
● appearance of isolated myoglobin isolation of gluten.
residue ● Isolates will be brought for testing
during our onsite laboratory.
Additional notes:
● Precipitating agent (NH4)2SO4 HYDROLYSIS OF INTACT PROTEINS
competes with the protein, ● ACID
displacing it, and attaches to water ● ALKALINE
afterwards. ● ENZYMATIC HYDROLYSIS
● The 20 amino acids are commonly

found as hydrolysis products of
proteins.
Isolation of Myoglobin
• It is a globular protein that is for O2 ● Hydrolysis can be carried out by
storage and transport in treating intact protein with acid, alkali
vertebrates, primarily in heart and skeletal or proteolytic enzymes.
muscles.
• It is a monomer made up of 153 aa
residues. • Principle involved in isolation:
Additional notes: ● 4M NaOH
● Multiple enzymes can be used ● 1M HCl
to hydrolyze proteins completely. ● 1M NaOH
○ E.g.: pepsin and ● Centrifuge tube
chymotrypsin ● Litmus paper
● 250-mL beaker
● Saturated protease solution/meat
HYDROLYSIS OF INTACT PROTEINS ● tenderizer
Isolated gluten, casein, albumin and
myoglobin ACID HYDROLYSIS OF INTACT PROTEINS
● 6 M HCl ● Cover the test tube with cotton and
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)

• Advantages: Little or no racemization
• Disadvantages: W is destroyed and is
● autoclave (15psi, 5 hrs).
converted to humin
● Take note the appearance of the
(black precipitate); partial destruction of S, T, C;
● mixture after autoclaving.
and hydrolysis of N to D & Q to E
● Add 10 mL of D.W. Transfer in a
250-mL beaker.
ALKALI HYDROLYSIS OF INTACT PROTEINS
● Neutralize the mixture with 1M NaOH.
● Cover the test tube with cotton and
● Use the neutralized mixture for
autoclave (15psi, 5 hrs)
● characterization and chromatography
● Take note the appearance of the
mixture after autoclaving. Add 10 mL of
D.W. Transfer in a 250-mL beaker. ●
Neutralize the mixture with 1M HCl.
● Use the neutralized mixture for
characterization and chromatography
Alkaline/Basic Hydrolysis – causes complete

hydrolysis of
Proteins.
● Isolated protein was treated with conc.
NaOH soln and was autoclaved at 15psi
for 5hrs.
● Advantages: W is stable
Hydrolysis of Proteins: Chemical Hydrolysis ● Disadvantages: Racemization of all
Acidic Hydrolysis – causes complete hydrolysis
amino acids; R decomposes to
of proteins.
• The isolated protein was treated with excess ornithine and urea; Partial destruction
6M HCl and of C,C-C, S, T
was autoclaved at 15psi for 5hrs.
Enzymatic Hydrolysis - proteolytic enzymes
Additional notes:
causes partial or
● Racemization - intramolecular
selective hydrolysis of polypeptide to yield
rearrangement
mixture
ENZYMATIC HYDROLYSIS OF INTACT

PROTEINS
● Incubate the mixture in a water bath
(35-40C) for 60 minutes.
● Allow the mixture to cool and proceed
to qualitative test and TLC
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)

of peptide fragments.
• Proteases/Peptidases – are enzymes that
hydrolyze peptide
bonds at specific sites
• Advantages: Amino acids are not affected; it
requires certain
temperature & pH conditions for optimum
activity of
enzymes. Hydrolysis is not complete.
Qualitative Color Reactions: Ninhydrin Test
● Add 50 uL of 0.1% Ninhydrin solution.

Mix well
● SHAKE, HEAT THE MIXTURE IN A
BOILING WATER BATH AND RECORD
THE COLOR OF THE SOLUTION.
GENERAL TEST FOR AMINO ACIDS WITH
QUALITATIVE COLOR REACTIONS: BIURET α-AMINO GROUP
TEST REAGENT: 1,2,3-INDANETRIONE
MONOHYDRATE OR
REAGENT/S: 2.5M NaOH and 0.1M TRIKETOHYDRINDENE HYDRATE AND
CuSO4 ETHANOL
● Add 400 uL of 2.5M NaOH. Mix PRINCIPLE: oxidative decarboxylation and
well deamination followed
● Add 20 uL of 0.1M CuSO4 by condensation
● SHAKE AND RECORD THE COLOR OF
THE SOLUTION
● POSITIVE RESULT: blue to blue-violet

GENERAL TEST FOR INTACT PROTEINS and
PROTEIN HYDROLYSATES
(TEST FOR PEPTIDE LINKAGES; AT LEAST UP Qualitative Color Reactions: Xanthorproteic
TO A TRIPEPTIDE) Test
● REAGENT: 2.5M NaOH and 0.1M
CuSO4 ● REAGENT: Conc. HNO3 and Conc.
● PRINCIPLE: formation of coordination NaOH
complex of copper and four nitrogen ● Add 10 drops of conc. HNO3. Mix well
atoms and observe the color change.
● POSITIVE RESULT: pink-violet to violet ● Slowly add 10 drops of Conc NaOH.
color
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)

● Mix and observe the color of the

solution with excess NaOH
GENERAL TEST FOR AROMATIC AMINO Additional notes:

ACIDS ● Micropipettes are not used for
● REAGENT: CONC. NITRIC ACID AND concentrated acids.
NaOH ○ Use serological pipettes
● PRINCIPLE: nitration of the aromatic instead.
rings via SEAr
Qualitative Color Reactions: Millon’s Test
● REAGENT: Millon’s reagent

● POSITIVE RESULT: Yellow Solution/ppt lemon
● Add 50 uL of Millon’s reagent. Mix well
yellow upon heating; orange
● RECORD THE COLOR OF THE Powder, oxalic acid and
SOLUTION Acetic acid) and SULFURIC ACID
● PRINCIPLE: reduction of oxalic acid to
DETECTS PHENOLIC RING CONTAINING glyoxylic acid and
AMINO ACID acid-catalyzed condensation of two
tryptophans with glyoxylic acid
● REAGENT: MILLON’S RGT. (Hg(NO3)2

and Hg(NO2)2)
● PRINCIPLE: Complexation (mercuration &
nitration)
● POSITIVE RESULT: old rose/reddish ● POSITIVE RESULT: pink to violet
brown/flesh interface
Qualitative Color Reactions: Hopkins-Cole
Test
● REAGENT: Hopkins-Cole reagent and
Conc. H2SO4
● Add 100 uL of HCR. Mix well Qualitative Color Reactions: Sakaguchi Test
● Incline the microcentrifuge tube and ● REAGENT: 10% NaOH and 0.2% a-naphthol
add slowly to the side of the tube 20 and NaOBr
drops of conc. H2SO4 ● Add 50 uL of 10% NaOH and 50 ul of
● DO NOT SHAKE THE MIXTURE. 0.02% napthol solution . Mix well
OBSERVE THE INTERFACE ● Mix well and let stand for 3 minutes.
DETECTS INDOLE GROUP CONTAINING ● Add 20 ul of 2% NaOBr.
AMINO ACID ● MIX AND RECORD the COLOR of the
● REAGENT: GLYOXYLIC ACID (Mg SOLUTION.
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)

● PRINCIPLE: Complexation reaction

DETECTS UN- OR MONOSUBSTITUTED
GUANIDINES
REAGENT: a-NAPTHOL, NaOBr, NaOH, Urea (to
stabilize the color
and destroy excess OBr- ions)
PRINCIPLE: complexation; base-catalyzed
condensation of anapthol with guanido group
of Arg
POSITIVE RESULT: RED TO RED ORANGE
COLOR
REAGENT: 30% NaOH and 5%
Pb(CH3COO)2
Add 50 uL of 30% NaOH and 20 uL 5%
Pb(CH3COO)2. Mix well
HEAT THE MIXTURE IN A BOILING
WATER BATH AND RECORD THE
COLOR OF THE SOLUTION
DETECTS SULFUR-CONTAINING AMINO ACIDS

REAGENT: LEAD ACETATE AND NAOH
PRINCIPLE: degradation and substitution
reaction to form PbS
POSITIVE RESULT: brown to black precipitate
Qualitative Color Reactions: Nitroprusside

Test
REAGENT: 3M NaOH and 2% Nitroprusside Qualitative Color Reactions: Amines Test
solution REAGENT: 20% NaOH
● Add 250 uL 3M NaoH then 250 ul of Add 500 uL of 20% NaOH. Mix well
● 2% nitroprusside solution. HEAT THE MIXTURE IN A BOILING
● RECORD THE COLOR OF THE WATER BATH AND TEST THE EVOLUTION
SOLUTION OF GAS by PLACING A RED LITMUS
PAPER OVER the mouth of the tube.
DETECTS CYSTEINE NOTE THE RESULT
● REAGENT: Na2Fe(CN)5NO in dil. NH3
and NaOH DETECTS PRIMARY, SEC AND TERT AMINES
POSITIVE RESULT: red coloration AND NITRILES
REAGENT: NaOH
PRINCIPLE: basic hydrolysis
Qualitative Color Reactions: FOHL’s Test
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)

(DIAZO RGT.) and 10%
Na2CO3
POSITIVE RESULT: red-blue litmus paper
Add 100 uL of Diazo reagent and
add 20 ul 10% Na2CO3
Qualitative Color Reactions: Pauly’s test
RECORD THE COLOR OF THE
(Ehrlich’s Diazo) Test
SOLUTION
REAGENT: 1% sulfanilic acid and 5%
NaNO2
TLC Plate
DETECTS TYR AND HIS • Chromatography chamber lined with Filter
REAGENT: DIAZO REAGENT (1% SULFANILIC paper
ACID • Capillary tubes
WITH 5% NaNO2) AND 10% Na2CO3 • Ruler
PRINCIPLE: formation of azo dye • Pencil
POSITIVE RESULT: RED COLORATION – His is
red to orange; Tyr is light orange STEPS IN TLC
PROTEINS
TLC Development
Chamber
1. Prepare a developing chamber.
2. Prepare the TLC plate.
3. Spot the plate with the sample.
4. Place inside the developing chamber. 5.
THIN LAYER
Spray with visualization (staining) reagent 6.
CHROMATOGRAPHY
Dry.
This techniques is used to separate and identify
7. Visualize the spots (using UV light) and mark
amino acid
with pencil.
constituents of the hydrolyzed protein samples.
8. Compute for Rf values.
Amino acids are
separated based on their polarities.
Composed of a stationary phase (TLC plates)
and a mobile phase
(solvent).
PRINCIPLE: difference in affinity of the solute
on the mobile and
stationary phase (“like dissolves like”)
Thin Layer Chromatography
• Amino Acid Standards
• 2% Trp, Arg, Pro, Cys, Ser, Asp, Tyr, His, Gly
and Ala
• Hydrolyzed protein samples
• 1-Butanol: Acetic Acid: Water (4:1:5) BAW
• 1% Ninhydrin Solution in Spray Bottle •
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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
• Visualization – chemical visualizing agent:
ninhydrin spray for 1.Alanine 0.30 0.46
amino acids and proteins
• Evaluation –comparing Rf values of sample 2.Aspargine 0.21 0.24
and standards 3.Aspartic 0.24 0.29
• Documentation – chromatogram Acid
AMINO ACIDS Rf Values
Silica gel Cellulose

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)

18.Tryptopha 0.61 • Bradford reagent
4.Arginine 0.16 n • Bovine Serum Albumin (BSA)
• Protein Sample (Albumin Intact
5.Cysteine 0.37 19.Tyrosine 0.55 protein) • 96-well microplate
6.Glutamine 0.25 20.Valine 0.44 • Micropipettes and tips
• Microplate reader
7.Glutamic 0.31
acid UV Spectrophotometry Method
0.32 0.41 0.29 0.35
8.Histidine 0.12 • Proteins will absorb ultraviolet
light at 280 nm. This is due to the
9.Isoleucine 0.53 0.32 0.73 0.26 0.24 0.71 0.60 tyrosine and tryptophan residues
in the protein. Quantitation is
10.Glycine 0.25 0.66
done by comparing the
11.Lysine 0.12 absorbance of the test solution
0.48 0.25 0.36 0.57 0.44 0.66 with a
12.Leucine 0.61
known standard.
A standard calibration curve of
13.Methionine 0.51 • Advantage
known protein sample will be
• The method is accurate, simple
14.Phenylal 0.62 utilized to compute for the
and highly sensitive up to
ani ne amount of proteins in the sample
microgram quantities.
using the absorbance with the
15.Proline 0.24 application of Beer-Lambert's law
Biuret Method
16.Serine 0.26
QUANTITATIVE PROTEIN
17.Threonine 0.30 ANALYSIS • Biuret Reagent
with a compound
QUANTITATIVE PROTEIN ANALYSIS The most producing a visible colored product which can
common method to determine protein be analyzed using
concentration spectrophotometer.
are; Biuret Test, Bradford Assay, Lowry Assay, •Cupric ions chelate (complex formation) with
and Bicinchoninic peptide bonds of proteins in alkaline medium
Acid (BCA) Test. produce a pink or violet color. The intensity of
Principle: the protein sample is made to react the
color is proportional to the number of peptide Biuret
bonds. The color is then compared with a reagent, and estimated colorimetrically @540
standard protein solution treated with the nm
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Biuret Method
•Advantage
● The biuret method is simple one-step
process, and is
● the most widely used method for
plasma protein estimations.
• Disadvantage
● The sensitivity of the method is
less
and is unsuitable for estimation
of
proteins in milligram or
microgram
quantities.
Bradford Method
• A simple and one of the most commonly used
assay
for total protein concentration.
• It is based on the proportional binding of
Coomassie
dye to proteins. The more proteins present,
more
dye will bind.
• It is a colorimetric assay. The color intensity of
the
The solution depends on the concentration of
protein.
•Principle involved: In an acidic medium,
proteins
bind to Coomassie dye due to hydrophobic and
electrostatic interactions.
•Λmax of dye = 465 to 595 nm
•Red solution Blue solution (stable up to 1 hr)
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UNIVERSITY OF SANTO TOMAS

FACULTY OF PHARMACY BATCH 2025 PHA6112

UNIVERSITY OF SANTO TOMAS

Glutelins – soluble in dilute solutions of acids

PROTEINS: Chemical Properties

UNIVERSITY OF SANTO TOMAS

UNIVERSITY OF SANTO TOMAS

ISOLATION OF ALBUMIN FROM SKIMMED

UNIVERSITY OF SANTO TOMAS

● It is a mixture of two proteins: glutenin

Additional notes: ISOLATION OF GLUTEN

UNIVERSITY OF SANTO TOMAS

● The 20 amino acids are commonly

UNIVERSITY OF SANTO TOMAS

Alkaline/Basic Hydrolysis – causes complete

ENZYMATIC HYDROLYSIS OF INTACT

UNIVERSITY OF SANTO TOMAS

● Add 50 uL of 0.1% Ninhydrin solution.

● POSITIVE RESULT: blue to blue-violet

UNIVERSITY OF SANTO TOMAS

● Mix and observe the color of the

GENERAL TEST FOR AROMATIC AMINO Additional notes:

Qualitative Color Reactions: Millon’s Test

● REAGENT: Millon’s reagent

● REAGENT: MILLON’S RGT. (Hg(NO3)2

UNIVERSITY OF SANTO TOMAS

● PRINCIPLE: Complexation reaction

DETECTS SULFUR-CONTAINING AMINO ACIDS

Qualitative Color Reactions: Nitroprusside

UNIVERSITY OF SANTO TOMAS

UNIVERSITY OF SANTO TOMAS

Silica gel Cellulose

UNIVERSITY OF SANTO TOMAS

UNIVERSITY OF SANTO TOMAS

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