Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Tryptophan
Determination of Tryptophan in Intact Proteins

Tryptophan in an intact protein can be determined spectrophotometrically [103, 128, 349], fluorometrically [337], or colorimetrically following its conversion into a colored derivative [11, 14, 334, 351]. Magnetic circular dichroism at 293 nm [13] and a scintillation assay using 3-diazo-1,2,4-'[5-¹⁴C]triazole [97] have been tested on a limited number of Proteins. At the picomolar level, tryptophan has been quantified via a modified Pictet-Spengler reaction: the mixture is boiled with potassium ferricyanide and formaldehyde, and the resulting 9-hydroxymethyl-β-carboline is measured spectrophotometrically or by reversed-phase HPLC [174].

Tryptophan is present in proteins in lower amounts compared to Other Amino Acids; consequently, its precise analysis enables an accurate Determination of the protein molecular weight.

3.7.3.1. Determination with 2-hydroxy-5-nitrobenzyl bromide [11]

Reagents

Urea, recrystallized from an ethanol-Water mixture.

2-Hydroxy-5-nitrobenzyl bromide (HNB-Br).

Trichloroacetic acid (TCA), 50%.

Procedure. The protein sample is incubated at 37°C for 16–20 h in 1 mL of 10 M urea acidified to pH 2.7 with concentrated HCl, cooled to room Temperature, and treated with 5 mg of HNB-Br in 0.1 mL of dry acetone added dropwise via a pipette submerged in the protein solution while stirring on a magnetic stirrer. The resulting precipitate (primarily the Hydrolysis product of the reagent) is removed by centrifugation.

The modified protein is applied to a Sephadex G-25 (coarse) Column (230X11 mm) equilibrated with 0.18 M acetic acid (pH 2.7) or 10 M urea (pH 2.7). Under these conditions, the protein remains in solution. Elution is carried out at a flow rate of 100 mL/h, collecting 1–4 mL fractions. The protein is precipitated with 50% TCA to a final concentration of 5%. If urea was used, the solution is diluted with a fivefold volume of water prior to adding TCA. The eluate is left at 4 °C for 30 min or overnight, and the resulting precipitate is separated by centrifugation, washed twice with 5 mL of an ethanol-HCl solution (2 mL of concentrated HCl + 98 mL of 95% ethanol). Finally, the pellet is dissolved in 1 mL of concentrated HCl, 0.1 mL of this solution is alkalinized with 2.5 M NaOH to pH 12, and the total volume is adjusted to 2.5 mL. The absorbance is measured at 410 nm (ε = 18,000). For Amino acid analysis, appropriate aliquots are hydrolyzed with 6 M HCl.