Question

A protein with 300 amino acids is to be sequenced. you are required to sequence it by adman degradation ​

Answer 1

Answer:

The protein of interest having been purified and its mass determined, the next analysis usually performed is to determine the protein's amino acid sequence, or primary structure. As stated previously (Section 3.2.1), a wealth of information about a protein's function and evolutionary history can often be obtained from the primary structure. Let us examine first how we can sequence a simple peptide, such as



The first step is to determine the amino acid composition of the peptide. The peptide is hydrolyzed into its constituent amino acids by heating it in 6 N HCl at 110°C for 24 hours. Amino acids in hydrolysates can be separated by ion-exchange chromatography on columns of sulfonated polystyrene. The identity of the amino acid is revealed by its elution volume, which is the volume of buffer used to remove the amino acid from the column (Figure 4.18), and quantified by reaction with ninhydrin. Amino acids treated with ninhydrin give an intense blue color, except for proline, which gives a yellow color because it contains a secondary amino group. The concentration of an amino acid in a solution, after heating with ninhydrin, is proportional to the optical absorbance of the solution. This technique can detect a microgram (10 nmol) of an amino acid, which is about the amount present in a thumbprint. As little as a nanogram (10 pmol) of an amino acid can be detected by replacing ninhydrin with fluorescamine, which reacts with the α-amino group to form a highly fluorescent product (Figure 4.19). A comparison of the chromatographic patterns of our sample hydrolysate with that of a standard mixture of amino acids would show that the amino acid composition of the peptide is





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