Method

Asparagine and glutamine are converted to aspartic acid and glutamic acid, respectively, during acid hydrolysis. Asparagine and aspartic acid residues are added and represented by Asx, while glutamine and glutamic acid residues are added and represented by Glx. Proteins/peptides can be reacted with bis(1,1-trifluoroacetoxy)iodobenzene (BTI) to convert the asparagine and glutamine residues to diaminopropionic acid and diaminobutyric acid residues, respectively, upon acid hydrolysis. These conversions allow the analyst to determine the asparagine and glutamine content of a protein/peptide in the presence of aspartic acid and glutamic acid residues.

Reducing solutions. Prepare and filter 3 solutions: a solution of 10 mM trifluoroacetic acid (Solution A), a solution of 5 M guanidine hydrochloride and 10 mM trifluoroacetic acid (Solution B), and a freshly prepared solution of dimethylformamide containing 36 mg of BTI per millilitre (Solution C).

Procedure. In a clean hydrolysis tube, transfer about 200 pg of the test sample, and add 2 ml of Solution A or Solution B and 2 ml of Solution C. Seal the hydrolysis tube in vacuo. Heat the sample at 60 °C for 4 h in the dark. The sample is then dialysed with water to remove the excess reagents. Extract the dialysed sample 3 times with equal volumes of butyl acetate, and then lyophilise. The protein can then be acid hydrolysed using previously described procedures. The a,P-diaminopropionic and a/y-diaminobutyric acid residues do not typically resolve from the lysine residues upon ion-exchange chromatography based on amino acid analysis. Therefore, when using ion-exchange as the mode of amino acid separation, the asparagine and glutamine contents are the quantitative difference in the aspartic acid and glutamic acid content assayed with underivatised and BTI-derivatised acid hydrolysis. The threonine, methionine, cysteine, tyrosine, and histidine assayed content can be altered by BTI derivatisation; a hydrolysis without BTI will have to be performed if the analyst is interested in the composition of these other amino acid residues of the protein/peptide.

METHODOLOGIES OF AMINO ACID ANALYSIS: GENERAL PRINCIPLES

Many amino acid analysis techniques exist, and the choice of any one technique often depends on the sensitivity required from the assay. In general, about one-half of the amino acid analysis techniques employed rely on the separation of the free amino acids by ion-exchange chromatography followed by post-column derivatisation (e.g., with ninhydrin or o-phthalaldehyde). Post-column derivatisation techniques can be used with samples that contain small amounts of buffer components, (such as salts and urea) and generally require between 5 pg and 10 pg of protein sample per analysis. The remaining amino acid techniques typically involve pre-column derivatisation of the free amino acids (e.g., phenyl isothiocyanate;

6-aminoquinolyl-N-hydroxysuccinimidyl carbamate or o-phthalaldehyde; (dimethylamino)azobenzenesulphonyl chloride; 9-fluorenylmethyl chloroformate; and

7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole) followed by reversed-phase HPLC. Pre-column derivatisation techniques are very sensitive and usually require between 0.5 pg and 1.0 pg of protein sample per analysis but may be influenced by buffer salts in the samples. Pre-column derivatisation techniques may also result in multiple derivatives of a given amino acid, which complicates the result interpretation. Post-column derivatisation techniques are generally influenced less by performance variation of the assay than pre-column derivatisation techniques.

The following methods may be used for quantitative amino acid analysis. Instruments and reagents for these procedures are available commercially. Furthermore, many modifications of these methodologies exist with different reagent preparations, reaction procedures, chromatographic systems, etc. Specific parameters may vary according to the exact equipment and procedure used. Many laboratories will use more than one amino acid analysis technique to exploit the advantages offered by each. In each of these methods, the analogue signal is visualised by means of a data acquisition system, and the peak areas are integrated for quantification purposes.

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