The recovery of tryptophan from hydrolysis of proteins with trifluoroacetic acid and hydrochloric acid
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A method has been developed for the determination of tryptophan in proteins. This technique modified the use of strong acid such as 6M HC1 containing mercapto-acetic acid (thioglycollic acid). Tri-fluoro acetic acid has been used to dilute concentrated HC1 and mercapto acetic acid was added to prevent the destruction of labile tryptophan during acid hydrolysis of lyophilized proteins in an evacuated sealed tube. Trifluoro acetic acid and hydrochloric acid (1:1) mixture serves several advanges: (1) The two reagents are miscible, (2) Trifluoro acetic acid is a good solvent for peptides and in strong acid, amino groups are protonated and hence not acetylated, (3) Trifluoro acetate can be easily removed and the point of attack is probably trifluro acetyl as well as carbonyl carbon. The activation of the β-carboxyl group of some amino acids could result in the formation of cyclic imide by displacement of tri-fluoro acetic acid. But this is doubtful, because if it occurs, there will be incomplete hydrolysis. The sample proteins were lyophilized and suspended in freshly prepared Hydrochloric acid diluted to 6M with trifluoro-acetic acid containing mercapto acetic acid. The tube was then placed in solid-carbon dioxide and ethanol. When frozen, the tube was evacuated to 0.1mm Hg and sealed under vacuum. Hydrolysis was conducted at a controlled temperature of 110° + 1°C for 24, 36 and 43 hours in a DRI-BLOCK heater. The excess solvent was removed on a rotatory vacuum evaporator connected to Paxman Cooling System, water bath at 37°C and oil pump pressure within 20 minutes. The residue was quantitively estimated using norleucine which is used as internal standard for J63LJLC-5AH Amino Acid Analyzer. The values of tryptophan and other amino acids obtained were close to the expected integral values and recoveries of all other amino acids were comparable to those observed after hydrolysis with 6M HC1 or 6M HC1 containing 4% mercapto acetic acid or p-toluene sulphonic acid. The method can also be used for protein containing about 5% (w/w) carbohydrate. The hydrolysate can also w be used in Miller's colorimetric method for the determination of tryptophan in purified proteins. The procedure, however, is not yet tested with feedingstuffs. Hence, a new method for the microanalysis of tryptophan by acid hydrolysis is proposed.
Thesis, MSc Master of Science
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