5-Methyltryptophan: An internal standard for tryptophan determination by ion-exchange chromatography

✍ Scribed by M. Wilkinson; G.A. Iacobucci; D.V. Myers

• Publisher: Elsevier Science
• Year: 1976
• Tongue: English
• Weight: 448 KB
• Volume: 70
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(76)90472-3

✦ Synopsis

The use of 5-methyltryptophan as an internal standard to facilitate tryptophan determination is described. Protein is hydrolyzed in the presence of 5-methyltryptophan for 18 hr at 120 ° in 5.0 N NaOH in the absence of oxygen and in the presence of starch or thiodiglycol as an antioxidant. Ion-exchange chromatography of the hydrolysate on Durrum DC-2 resin using pH 5.43 citrate (0.175 N Na ÷) completely resolved tryptophan and 5-methyltryptophan from one another, other amino acids, and artifacts of the alkaline hydrolysate. The chromatographic conditions and stability of tryptophan and 5-methyltryptophan were established initially by demonstrating quantitative recovery of both amino acids that had been added prior to hydrolysis of ribonuclease A, a protein devoid of tryptophan. The tryptophan content of several well-characterized proteins was determined, and the results, after correction to 100% recovery of 5-methyltryptophan, agreed well with values obtained by established procedures.