Spectroscopic Determination of Tryptophan and Tyrosine in Proteins^*

Abstract

A rapid method for the determination of tryptophan in proteins is presented. It is based on absorbance measurements at 288 and 280 mp of the protein dissolved in 6 M guanidine hydrochloride. Blocked tryptophanyl (N-acetyl-L-tryptophanamide) and tyrosyl (glycyl-L-tyrosylglycine) compounds were selected as C urrent methods of protein amino acid analysis do not give quantitative values for tryptophan and consequently the amino acid compositions, which are otherwise complete, fail to report tryptophan values. The principal reason for this situation is that the standard procedure of protein hydrolysis in strong acid results in the destruction of tryptophan (Hill, 1965). Therefore a second procedure is required to measure tryptophan. Alkaline hydrolysis is less destructive but does not give quantitative recoveries generally (Spies and Chambers, 1949). Enzymatic hydrolysis of proteins can give quantitative yields of tryptophan but this method may not be generally valid (Hill and Schmidt, 1962).The hydrolytic problem can be circumvented by measuring tryptophan in the intact protein. A chemical method has been developed which has not been exploited adequately Chambers, 1948, 1949). On the other hand, considerable effort has been expended in developing absorption spectroscopic procedures to measure tryptophan and tyrosine in unhydrolyzed proteins. Holiday (1936) and Goodwin and Morton (1946) have measured the absorption of proteins in 0.1 M NaOH and computed their tryptophan and tyrosine contents based on comparison with the absorption of the two amino acids. A modification of these techniques has been presented by Bencze and Schmid (1957). The preceding three methods do not give quantitative results. The behavior of the chromophores has not been normalized and the two models, i.e., tryptophan and tyrosine, are not completely adequate. A procedure is suggested in this report which strongly reduces or eliminates these interactions, normalizes their absorption, and consequently permits a more precise analysis of tryptophan and tyrosine in proteins.

Isolation and characterization of the cyanogen bromide fragments from human lactotransferrin

“…* The number of serine and threonine were extrapoled to zero time, while that of leucine and isoleucine were determined after 70 hr hydrolysis. ** Edelhoch method [21]. *** ND: not determined.…”

Structural characterization of the tumor suppressor p16, an ankyrin‐like repeat protein

“…We determined protein concentrations by two or more of the following methods: absorbance at 280 nm in 6 M guanidine-HC1, IO mM MOPS, pH 7.5 using an extinction coefficient of 13,940 (Edelhoch, 1967), amino acid analysis, or with the Coomassie Plus protein assay using bovine serum albumin as the standard.…”

Cis proline mutants of ribonuclease A. I. thermal stability

“…The concentrations of the mutant proteins were determined either by amino acid analysis or by tyrosine absorbance at 278 nm in 6 M GdmC1, 0.05 M sodium phosphate, pH 6.5, using a molar absorbance based on the number of tyrosine residues (Edelhoch, 1967). Enzymatic activities were assayed with the substrate 2',3'-cCMP (0.3 mg/mL) in 0.05 M Na cacodylate, pH 7.0, at 18 "C (Crook et al, 1960).…”