PROTEINS
(Isolation, Hydrolysis,
Qualitative Tests and
Quantitative Determination)
ISOLATION
CASEIN
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main protein in milk
exists as the Ca salt
phosphoprotein
mixture of min of 3 similar proteins (-, - &  casein)
80% of protein present in milk
contains the essential amino acids (V P H MATILL)
isolated at isoelectric pH (pI), least soluble
(isoelectric precipitation)
accomplished by addition of dilute acid
net charge at pI=0
HYDROLYSIS
• bond cleavage of labile bonds simultaneous
with the addition of water
O
H2 O
X
O
+
HX
OH
• needed to break amide bonds in intact
proteins to produce amino acids
Types of Protein Hydrolysis
Acid hydrolysis – catalyzed by strong
acids such as H2SO4, HCl, HNO3,
HClO4, etc. (15 psi/5 hrs.)
• total hydrolysis
• does not promote racemization of a-C
configuration
• Trp is destroyed and converted to humin
(black pigment)
• Thr and Ser are destroyed
• Asn and Gln are converted to Asp and Glu
Base/Alkaline Hydrolysis – uses
strong bases such Ba(OH)2,
NaOH, KOH, etc. (15 psi/5hrs.)
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total hydrolysis
Trp is not destroyed
promotes racemization
Thr and Cys are lost
Arg is destroyed and converted to urea
& ornithine
Enzymatic hydrolysis – partial
cleavage/hydrolysis
• regioselective and/ stereoselective
• cleaves specific linkages of selected
types of amino acid groups (i.e.
carboxypeptidase A for aromatic AA’s)
QUALITATIVE
CHEMICAL TESTS
Biuret Test – general test for
intact proteins and protein
hydrolyzates (at least a
tripeptide!)
O
• named after the compound, biuret
H2 N
O
N
N H2
H
• reagents: CuSO4 solution and dilute NaOH
• positive result: formation of pink to violet to blue
color
• principle: complexation of Cu+2 with amide N atoms
• NO reaction with dipeptides, urea, coagulated
proteins and amino acids (except serine and
threonine)
O
O
H N
Cu
R
H
O
N H
+ 2
N
O
H
R
H
N
H
Ninhydrin Test – general test
for compounds with free a –
amino groups
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one of the most sensitive color reactions known
reagent/s: ninhydrin (1,2,3 - indanetrione monohydrate)
in ethanol
positive result: blue to blue violet color
principle: oxidative deamination and decarboxylation;
reduction of ninhydrin
Proline, hydroxyproline, and 2-, 3-, and 4-aminobenzoic
acids fail to give a blue color but produce a yellow color
instead
ammonium salts give a positive test. Some amines,
such as aniline, yield orange to red colors, which is a
negative test
O
O
O
OH
OH
+
R
H
+
OH
RCHO
+
CO 2
+
NH3
OH
NH2
O
O
O
O
OH
OH
O
+
2 NH3
+
H
+
NH4 O
O
HO
O
+
-
N
O
O
H2O
Xanthoproteic Test – general test for
aromatic amino acids such as
tryptophan, phenylalanine, histidine
and tyrosine
• presence of electron donating substituents
enhances reaction rate
• reagents: conc. HNO3 and conc. NaOH
(neutralize excess acid)
• positive results: formation of yellow
precipitate and after addition of excess
NaOH (alkaline), an orange precipitate forms
• principle involved: nitration of aromatic
rings (i.e. indole in tryptophan!) via
electrophilic aromatic substitution
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O
O
N H3
HNO 3
HO
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O
N H3
N
O
N H3
x' cess NaOH
O
O
OH
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O
N
O
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O
-
Hopkins-Cole Test – detects the
presence of indole group in
tryptophan
• reagents: magnesium, oxalic acid and
conc. H2SO4
• positive result: pink to violet interface
• principle: reduction of oxalic acid to
glyoxilic acid & acid-catalyzed
condensation of 2 tryptophans with
glyoxilic acid
OH
OH
O
O
N H3
O
-
N
H
O
+
O
Mg
O
O
H3 N
OH
H
H
-
O
+
OH
H
O
N
H
N
O
-
O
N H3
Sakaguchi Test – specific for
arginine (guanido group)
• reagents: -napthol, NaOH and NaOBr
(and urea to stabilize color and destroy
excess OBr- anions)
• positive result: red to red-orange
color
• principle: base-catalyzed condensation
of -napthol with the guanido group of
arginine
H3 N
H3 N
+
H
H
O
O
O
N H2
-
OH
-
NH
N
+
O
+ 2
OH
-
N
N
H
OH
H
N
O
QUANTITATIVE
DETERMINATION
OF PROTEINS
Bradford Assay – simple, fast,
inexpensive, highly sensitive
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uses the Coomassie Brilliant Blue G-250 dye reagent ( binds
electrostatically with arginine residues in anionic form and by pistacking interactions with aromatic AA’s)
read at 595 nm (UV spectrophotometer)
intensity of color (measured by absorbance) is directly proportional
to the concentration of protein (Beer’s Law)
A = bc
unknown concentration is measured using linear regression analysis
y = mx + b
where: y = measured absorbance
m = slope
x = concentration of unknown
b = y-intercept
for standard protein preparations, use C1V1=C2V2 when dilutions
are done on standard solutions.
H 3 CCH 2
SO 3
N
-
H 3 CH 2 CO
N
N
H
CH 2 CH 3
SO 3 N a