Explain how scientists determine the kinds and numbers of amino acids in a protein?

Question

Explain how scientists determine the kinds and numbers of amino acids in a protein?

Answer 1

you first extract the amino acids by destroyying the peptide binds via hydrolysis then testing the hydrolyzate through color reactions which would determine the presence of specific amino acids :D

i hope i was able to answer your question :D

Answer 2

Determining amino acid composition

It is often desirable to know the unordered amino acid composition of a protein prior to attempting to find the ordered sequence, as this knowledge can be used to facilitate the discovery of errors in the sequencing process or to distinguish between ambiguous results. Knowledge of the frequency of certain amino acids may also be used to choose which protease to use for digestion of the protein. A generalised method for doing this is as follows:

Hydrolyse a known quantity of protein into its constituent amino acids.

Separate the amino acids in some way.

[edit] Hydrolysis

Hydrolysis is done by heating a sample of the protein in 6 Molar hydrochloric acid to 100-110 degrees Celsius 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 cystine) 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 - 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.

[edit] 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 respective isoelectric 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.

[edit] 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 blue 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.

Answer 3

they use special techniques. There is x-ray crystalography, of the protein is small enough NMR. There is also a way to use enzyme which cut proteins in very specific manner. Carboxypeptidase, chemotrypsin, trypsin, Edman Degredation, and sanger reactions.