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-