Practical Protein Chemistry - A. Darbre 1989

Analytical methods
Acetyl and formyl groups

Following the initial report on the isolation of an N-acetylated peptide from the N-terminal region of tobacco mosaic virus protein [273], a significant number of Proteins with blocked α-amino groups have been discovered. N-Acetylated residues of Alanine, aspartic acid, Glycine, Serine, and Threonine have been identified in proteins isolated from various sources [47, 81]; see also the reviews [2 and 406].

Protein chain synthesis on the ribosome is initiated with N-formylmethionine, while formylvaline and formylglycine are located at the N-termini of gramicidin A [325] and melittin [203]. Spontaneous N-formylation of L-Lysine residues by formaldehyde has also been described [371].

Various Methods have been employed to determine The Nature of the acylating residue in proteins. For instance, the acetyl group has been determined using enzymatic methods [135, 201, 204, 319, 346, 354], colorimetrically as a complex with iron hydroxamate [227], by steam distillation followed by titration [7, 183], and identified in the form of acetohydrazide and 1-acetyl-2-DNP-hydrazide [301] or 1-acetyl-2-dansylhydrazide [329]. The application of enzymatic methods requires highly purified enzyme preparations and typically demands substantial amounts of substrate (100 nmol of acetate per sample), although a Procedure for determining acetate at the level of 3–12 nmol/mL with fluorescence detection has been described [135].

The formyl group was determined colorimetrically (according to Barker and Summerson) following reaction with 2-thiobarbituric acid, which leads to The formation of a chromophoric group with λ_mах = 450 nm (phosphates and acetic acid do not interfere with the determination). The absorbance-to-concentration dependence is linear in the range of 1—10 mmol, but the analytical sensitivity is low [113].

If the N-terminal region of a protein can be isolated as a short peptide, the content of the acetylated amino acid within it can be determined after Hydrolysis using chromatographic or electrophoretic analysis [414]. To establish the Nature of the blocking residue alongside the Amino Acid Sequence of the peptide, the direct mass spectrometric method is the most reliable (requiring ~50 pmol of peptide for analysis) (Ch. 19), [6].

Several Procedures have been developed for the determination of acetyl groups in proteins using GLC [45, 154, 164, 332, 389, 390] and pyrolysis [402]. However, Gas-Liquid Chromatography can be accompanied by acetic acid tailing on the Column, ghost peaks, and a low detector response.

It has been proposed to cleave Acetyl and formyl groups by alkaline hydrolysis of the protein [54], since acid hydrolysis generates free acetic and formic acids, making their subsequent losses inevitable. Following alkaline hydrolysis, the salts of these acids are converted into phenacyl esters catalyzed by crown ethers (dicyclohexyl-18-crown-6). For the detection of phenacyl esters using a flame ionization detector, the acyl group content in the sample must be sufficiently high (~20 nmol). Fully exploiting the sensitivity of the GLC method remains a challenge to this day, because under standard conditions only a small fraction of the resulting sample derivative can be injected into the column. In the described case, 1—2 nmol of the phenacyl ester was introduced into the column. Pentafluorobenzyl (PFB) esters are more efficient in this regard, offering higher detection sensitivity due to the feasibility of using an electron-capture detector.