PROTEINS(Isolation, Hydrolysis, Qualitative Tests and
Quantitative Determination)
ISOLATION
CASEIN • 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
• needed to break amide bonds in intact proteins to produce amino acids
O
XH2O
O
OH+ HX
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.)• 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!)
• named after the compound, biuret
• 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)
H2N
O
NH
NH2
O
NOH
HN
R
H
OCu+ 2O
NH RH
O N H
Ninhydrin Test – general test for compounds with free a – amino groups
• 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
OH
OH+R
NH2
O
OH
O
O
H
OH+ CO2RCHO NH3++
O
O
OH
OH
O
O
OH
H+ NH3 +
ON
O
O-
O2 OH2+NH4
+
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
NH3
O-O
HNO3 x'cess NaOHNH3
OHO
NO
O -
NH3
O-O
NO
O -
OH-
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
N
NH3
O-O
H
+ H
O
O
OH
N
H3N
O -O
H
H
N
NH3-O
O
OHOH+
O
OH
O
OH
Mg
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
H3N+ HO -
O
N NH2
NH
H
+
OH
OH-
H3N+ HO -
O
NH
NN
O
OH
2
QUANTITATIVE DETERMINATION
OF PROTEINS
Bradford Assay – simple, fast, inexpensive, highly
sensitive• uses the Coomassie Brilliant Blue G-250 dye reagent ( binds
electrostatically with arginine residues in anionic form and by pi-stacking 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.
NH3CCH2
N NH
CH2CH3
H3CH2CO
SO 3Na
SO 3-
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