Amino Acid + Amino Acid + Amino Acid Peptide linkage

23 Pages·2013·2.02 MB·English

Amino Acid + Amino Acid + Amino Acid Peptide linkage
Proteins (many peptide linkages)
Proteins Amino Acids
CCaasseeiinn CCyysstteeiinnee
Gelatin Arginine
Albumin Tryptophan
Peptone Tyrosine
Tyrosine
Cysteine
Arginine Tryptophan
Tests to be done:
(cid:1)
Ninhydrin’s test (specific to all Proteins & Amino acids)
(cid:1)
Biuret’s test (specific to Proteins)
(cid:1)(cid:1)
SSaakkaagguucchhii’’ss tteesstt ((ssppeecciiffiicc ttoo AArrggiinniinnee))
(cid:1)
Nitropruside’s test (specific to Cysteine)
(cid:1)
Millon’s test (specific to Tyrosine)
(cid:1)
Hopkins-Cole’s test (specific to Tryptophan)
Ninhydrin’s test
Ninhydrin reaction
Principle:
Ninhydrin is Specific for Amino Acids & Proteins – to differentiate between
Carbohydrates (-ve) and Amino Acids & Proteins (+ve).
Ninhydrin Reacts with α-amino acids (–NH ) in proteins giving a purple colored
2
complex, except Proline and hydroxy proline gives yellow color(no –NH ).
2
Ninhydrin is most commonly used as a forensic chemical to detect
“fingerprints”, as amines left over from proteins sloughed off in
fingerprints react with ninhydrin giving a characteristic purple color.
Procedure & observation:
− To 1 mL amino acid solution in a test tube, add 2 drops of ninhydrin reagent.
− On cold and observe the formation of a purple color, OR
- Put in a boiling water bath and observe the formation of a purple color.
NNiinnhhyyddrriinn’’ss
tteesstt
Few drops of B
Mix Mix Heat
22--33 mmiinn
1ml of protein
C
Mix
solution
D
Result
Unknown A Proteins or Amino Acids & Carbohydrates
Reagent B Drops of Ninhydrin reagent
Observation C Dark purple colour on heat Proteins (free –NH )
2
D Purple-Violet colour or ppt on cold Amino Acids(free –NH )
2
D No reaction Carbohydrates
Biuret’s test
Principle:
Biuret test is Specific for Proteins – To differentiate between Proteins (+ve) and
Amino Acids (-ve). The biuret reagent (copper sulfate in a strong base) reacts with
peptide bonds in proteins to form a blue to violet complex known as the “Biuret
complex”.
Two peptide bonds at least are required for the formation of this complex.
Procedure & observation:
− To 1 mL of protein solution in a test tube, add 2 mL of 10% sodium
hydroxide solution and 2 drops of 1% copper sulfate solution.
− Mix well; a violet color is obtained with albumin, casein & gelatin and a pinkish
violet color with peptone.
Biuret’s test
2 drops of B
1ml of NaOH
Mix Mix
1ml of protein
C
solution
Result
Unknown A Proteins or Amino Acids
Reagent B 2 ml NaOH, then 2 drops of
CuSO4 (1%)
Observation C Violet colour Proteins (reacts with Di-peptide bonds)
C No change in colour (Blue) Amino Acids (No di-peptide linkage)