Answer to Question #343922 in Biochemistry for Vanesa

Question

Proteins are dynamic macromolecules. To study proteins a scientist needs to understand their chemical and physical properties. You are a scientist working in an analytical laboratory, your job description is to analyse proteins. A crude sample containing a protein is to be analysed. Discuss all steps and techniques that can be employed in order to isolate, characterise, and determine its amino acid sequence.

Answer 1

Determining amino acid composition[edit]

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. The misincorporation of low levels of non-standard amino acids (e.g. norleucine) into proteins may also be determined.^[1] A generalized method often referred to as amino acid analysis^[2] for determining amino acid frequency is as follows:

1. Hydrolyse a known quantity of protein into its constituent amino acids.
2. Separate and quantify the amino acids in some way.

Hydrolysis[edit]

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.

Separation and quantitation[edit]

The amino acids can be separated by ion-exchange chromatography then derivatized to facilitate their detection. More commonly, the amino acids are derivatized then resolved by reversed phase HPLC.

An example of the ion-exchange chromatography 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 are eluted when the pH reaches their respective isoelectric points. 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, fluorescent derivatives can be formed using reagents such as ortho-phthaldehyde (OPA) or fluorescamine.

Pre-column derivatization may use the Edman reagent to produce a derivative that is detected by UV light. Greater sensitivity is achieved using a reagent that generates a fluorescent derivative. The derivatized amino acids are subjected to reversed phase chromatography, typically using a C8 or C18 silica column and an optimised elution gradient. The eluting amino acids are detected using a UV or fluorescence detector and the peak areas compared with those for derivatised standards in order to quantify each amino acid in the sample.