Determine Structure of Undigested Protein

 

 

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Digestion of proteins can be carried out using 2 methods:

 

Acid-Base Hydrolysis Enzyme Cleavage
  • Non-selective
  • Overlapping fragments
  • Selective
  • No overlapping fragments
  • Require 2 sets of data from different enzymes to elucidate structure

 

Approach

 

Acid-Base Hydrolysis Enzyme Cleavage
  • Spot repetition of residues in fragments
  • Determine terminal residues; common in 2 sets of data
  • Spot repetition between the sets of data

 

Example (Hydrolysis):

 

Partial hydrolysis of a polypeptide produced the following fragments:

 

asp – lys – gly

val – phe – asp

lys – val – phe

gly – phe – lys

 

Determine the structure of the undigested polypeptide.

 

Spot repetition of residues in fragments

 

Structure: val – phe – asp – lys – gly – phe – lys – val – phe

 

Example (Enzyme):

 

A polypeptide was digested with 2 different enzymes. For each enzyme, the following fragments were obtained.

 

Enzyme 1

Enzyme 2

asp – lys – gly – phe

lys – val – arg

val – phe

gly – phe – lys

val – arg

val – phe – asp – lys

 

Determine the structure of the undigested polypeptide.

 

Step 1: Determine terminal residues; common in 2 sets of data

→ left terminal: val – phe

 

Step 2: Spot repetition between the sets of data

Green: From Enzyme 2, we can determine:

val – phe is followed asp – lys

 

Blue: From Enzyme 1, we can determine:

val – phe – asp – lys is followed by gly – phe

 

Yellow: From Enzyme 2, we can determine:

val – phe – asp – lys – gly – phe is followed by lys

 

Purple: From Enzyme 1, we can determine:

val – phe – asp – lys – gly – phe – lys is followed by val – phe

 

Structure: val – phe – asp – lys – gly – phe – lys – val – phe