1. PROTEINS
(Isolation, Hydrolysis, Qualitative Tests and Quantitative Determination)

2. ISOLATION

3. 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

4. HYDROLYSIS
bond cleavage of labile bonds simultaneous with the addition of water
needed to break amide bonds in intact proteins to produce amino acids

5. Types of Protein Hydrolysis

6. 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

7. 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

8. 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)

9. QUALITATIVE CHEMICAL TESTS

10. 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)

12. 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

14. 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

16. 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

18. 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

20. QUANTITATIVE
DETERMINATION
OF PROTEINS

21. 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.