PRIMARY PROTEIN

ROLL NO. : 87;88

 INTRODUCTION

• Proteins are the polymers of L- alpha amino acids

• The structure of protein is complex and divided into 4 levels :
• 1)Primary structure
• 2) Secondary structure
• 3) Tertiary structure
• 4) Quaternary structure
 BONDS IN PRIMARY STRUCTURE OF PROTEIN

1)Peptide bond 2)Sulfide bond

For example : For example :
 DETERMINATION OF PRIMARY STRUCTURE

• The primary protein comprises the identification of constituent amino acids with
regard to their quantity , quality and sequence in the protein structure.
• A pure sample of protein or polypeptide is essential for the determination of
primary structure which involves 3 stages :
1. Determination of amino acid composition
2. Degradation of protein or polypeptide into smaller fragments
3. Determination of the amino acid sequence.
 DETERMINATION OF AMINO ACID COMPOSITION

• The protein or polypeptide is completely hydrolysed to liberate the amino acids which
are quantitatively estimated.
• The hydrolysis may be carried out either by acid or alkali treatment or by enzyme
hydrolysis.
• Treatment with enzymes, however results in smaller peptides rather than amino acids
• Pronase is a mixture of non-specific proteolytic enzymes that causes complete
hydrolysis of proteins.
• Separation and estimation of amino acids:The mixture of amino acids liberated by
protein hydrolysis can be determined by chromatographic techniques.
DEGRADATION OF PROTEIN OR POLYPEPTIDE
INTO SMALLER FRAGMENTS
• Protein is a large molecule which is sometimes composed of
individual polypeptide chains. Separation of polypeptides is essential
before degradation.
• Liberation of polypeptides
• Number of polypeptides
• Breakdown of polypeptides into fragments : Polypeptides are degraded into smaller
peptides by enzymatic or chemical methods.
• Enzymatic cleavage : The proteolytic enzymes such as trypsin, chymotrypsin, pepsin
and elastase exhibit specificity in cleaving the peptide bonds . Among these enzymes,
trypsin is most commonly used. It hydrolyses the peptide bonds containing lysine or
arginine on the carbonyl ( C O) side of peptide linkage.
• Chemical cleavage : Cyanogen bromide (CNBr) is commonly used to split
polypeptides into smaller fragments. CNBr specifically splits peptide bonds, the
carbonyl side of which is contributed by the amino acid methionine.
DETERMINATION OF AMINO ACID SEQUENCE

• Using Sanger’s reagent

• DNFB
• Using Edman’s reagent
THANK YOU