The mixture of amino acids resulting from acid hydrolysis of a protein is separa

Question

The mixture of amino acids resulting from acid hydrolysis of a protein is separated and quantitated by:
A. Size exclusion chromatography and post column quantitation by the ninhydrin reaction
B. Ion exchange chromatography and post column quantitation by the ninhydrin reaction
C. Size exclusion chromatography with pre-column derivatization by phenyl isothiocyanate
D. Reversed phase HPLC and pre-column derivatization by phenylisothiocyanate

Which of the above statements are correct?

Explanation / Answer

B & D are the perfect answers.

Explanation for understnading:

sequncing analysis of protien:

step1 :Hydrolysis

Hydrolysis is done by heating a sample of the protein in 6 M hydrochloric acid to 100–110 °C for 24 hours or longer. Proteins with many bulky hydrophobic groups may require longer heating periods. However, these conditions are so vigorous that some amino acids (serine, threonine, tyrosine, tryptophan, glutamine, and cysteine) are degraded. To circumvent this problem, Biochemistry Online suggests heating separate samples for different times, analysing each resulting solution, and extrapolating back to zero hydrolysis time. Rastall suggests a variety of reagents to prevent or reduce degradation, such as thiol reagents or phenol to protect tryptophan and tyrosine from attack by chlorine, and pre-oxidising cysteine. He also suggests measuring the quantity of ammonia evolved to determine the extent of amide hydrolysis.

step 2:Separation

The amino acids can be separated by ion-exchange chromatography or hydrophobic interaction chromatography. An example of the former is given by the NTRC using sulfonated polystyrene as a matrix, adding the amino acids in acid solution and passing a buffer of steadily increasing pH through the column. Amino acids will be eluted when the pH reaches their respectiveisoelectric points. The latter technique may be employed through the use of reversed phase chromatography. Many commercially available C8 and C18 silica columns have demonstrated successful separation of amino acids in solution in less than 40 minutes through the use of an optimised elution gradient.

step 3:Quantitative analysis

Once the amino acids have been separated, their respective quantities are determined by adding a reagent that will form a coloured derivative. If the amounts of amino acids are in excess of 10 nmol, ninhydrin can be used for this; it gives a yellow colour when reacted with proline, and a vivid purple with other amino acids. The concentration of amino acid is proportional to the absorbance of the resulting solution. With very small quantities, down to 10 pmol, fluorescamine can be used as a marker: This forms a fluorescent derivative on reacting with an amino acid.

N-terminal amino acid analysis

Determining which amino acid forms the N-terminus of a peptide chain is useful for two reasons: to aid the ordering of individual peptide fragments' sequences into a whole chain, and because the first round of Edman degradation is often contaminated by impurities and therefore does not give an accurate determination of the N-terminal amino acid. A generalised method for N-terminal amino acid analysis follows:

There are many different reagents which can be used to label terminal amino acids. They all react with amine groups and will therefore also bind to amine groups in the side chains of amino acids such as lysine - for this reason it is necessary to be careful in interpreting chromatograms to ensure that the right spot is chosen. Two of the more common reagents are Sanger's reagent (1-fluoro-2,4-dinitrobenzene) and dansyl derivatives such as dansyl chloride. Phenylisothiocyanate, the reagent for the Edman degradation, can also be used. The same questions apply here as in the determination of amino acid composition, with the exception that no stain is needed, as the reagents produce coloured derivatives and only qualitative analysis is required. So the amino acid does not have to be eluted from the chromatography column, just compared with a standard. Another consideration to take into account is that, since any amine groups will have reacted with the labelling reagent, ion exchange chromatography cannot be used, and thin layer chromatography or high-pressure liquid chromatography should be used instead.

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