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Amino Acid Composition in Proteins
Once we purify the protein of interest, what should we do next? If we do not know anything about the protein, then the next logical step is to determine the composition of the protein. That is, we need to determine the types of amino acids and the number of each type in that protein. To do this, we must first expose the protein to a 6.0 M solution of hydrochloric acid and heat it to 110 degrees Celsius for about 24 hours. This will hydrolyze the peptide bonds and break down the protein into its individual constituent amino acids. The next step is to actually isolate the different amino acids and we can do this by using ion-exchange chromatography. Once we pour the solution into the column, the amino acids will then bind to the gel beads in that column with different affinities. They have different affinities because the amino acids contain different side chain groups. We can then use a buffer solution (i.e. sodium citrate) of increasing pH to elute the amino acids at different rates. Since it takes different volumes of buffer to actually elute the amino acid, we can elute and collect the amino acids one at a time. We can then compare the volume and pH of buffer that was needed to elute the particular amino acid to the standard textbook values and this will tell us what amino acid we are dealing with. Finally, to count how many amino acids we have in our sample, we can expose each solution containing a specific amino acid to ninhydrin. Ninhydrin reacts with the amino acid to produce a molecule that gives off a deep blue or deep purple color. This molecule can absorb light at amounts that is proportional to its concentration. Therefore, if we measure the light absorbance of each solution, that will tell us the relative concentrations of each amino acid in the protein.
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