Amino acid composition   In order to determine types of amino acids and their molar ratio in a polypeptide chain, complete hydrolysis may be carried out by heating a protein with 6N HCI at 110^o in an evacuated sealed tube for 6 to 48 hours. The resulting mixture, from which excess of HCI is removed, is called protein hydeolyzate containing a mixture of all constituent amino acids. Acid treatment cleaves peptide bonds to free the modified, e.g., tryptophan is destroyed while glutamine and asparagine are modified to their respective acids. However, amino acids thus obtained may be quantitatively determined by column chromatography or amino acid analyzer. The total amino acid content of β-lactogloulin (a milk protein) was determined in this manner. Proteins can be hydrolyzed by boiling with alkali, but this method is unsuitable since it causes racemization of most and destruction of some amino acids. The amino acids that are destroyed are cysteine,serine, threonine and cystine.

cleaving sites of some proteolytic enzymes used in partial hydrolysis

Partial hydrolysis   Sequencing of amino is done by partial hydrolysis of polypeptides. This is achieved by using specific enzymes which cleave the polypeptides at specific sites to yield smaller peptide fragments . Some enzymes and their specific sites of action are given. It is advisable to carry out partial hydrolysis simultaneously by two enzymes so that overlapping peptide fragments are obtained. The fragments so formed are isolated by methods, such as gel filtration and ion exchange chromatography. 

Determination of N-terminal Amino acid

1.    Sanger,s method  The N-terminal amino acid is identified by the reaction with 1-fluoro-2, 4-dinitrobenzene (FDNB) yielding dinitrophenyl residue of the N-terminal amino acid. The DNP-peptide is then hydrolyzed by 6N HCI so that all peptide bonds are cleaved leaving the DNP-derivatives. Since this method involves hydrolysis of peptide, it cannot be used for stepwise degradation