Identification of photochemical products of amino acids, peptides, and proteins in on‐line, postcolumn photolytic derivatization detection by HPLC‐electrochemistry

Abstract

ABSTMCTIn previous studies we have developed a method for the electrochemical detection of aromatic amino acids, sulfur-containing amino acids, peptides, and proteins in HPLC by using photolytic derivatization approaches. Upon irradlation, nonelectroactive (or even inherently electroactive) amino acids and peptides can be converted to electroactive (or more electroactive) compounds, in an on-line, postcolumn manner, which can be detected electrochemically in HPLC. In the case of proteins, the same method can be used to change the electroactivity of proteins photochemically. It is important to identify these photochemically converted compounds, especially those which are electroactive, in order to understand the detection mechanisms of the method. In this paper the results of identification of electroactive photoproducts for aromatic amino acids, peptides, and proteins are presented. Mainly, these studies were carried out by HPLC and direct electrochemical detection of off-line photolyzed analytes. m Y WORDS:Photolytic derivatization, proteins, peptides, amperometric detection.In the development of on-line, postcolumn electrochemical detection (EC) of aromatic amino acids, peptides, and proteins, a photolytic derivatization approach was used to convert nonelectroactive (or even mherently eleceoactive) amino acids and peptides into electroactive (or more electroactive) compounds, in an on-line, postcolumn manner, which could then be detected electrochemically in high performance liquid chromatography (HPLC) 11-21, In the case of proteins, the electroactivity could be increased upon irradiation [31. However, the exact photoproducts of these analytes were not identified in the previous studies. The identification of these photochemically converted compounds, especially those which are electroactive, is important to our understanding of the detection mechanism(s) of the overall method. In this paper the results of attempts to identify the electroactive photoproducts for aromatic or sulfur containing amino acids, peptides, and proteins are presented. Mainly, such 'Current Address: Roche Biomedical Laboratories, Inc., 1 Roche Drive, PO Box 500, Raritan NJ 08869 USA T o whom correspondence should be addressed. studies were carried out by HPLC and direct electrochemical detection of off-line photolyzed analytes.Studies on the photochemistry of amino acids, peptides, and proteins, especially aromatic and sulfurcontaining amino acids, have been heavily reported in the literature 14-71, Both radiolysis and photolysis were used for these irradiations. Different irradiation conditions, including wavelength of UV light, pH, concentration, and under N, or 0,, were employed. In most cases the final photochemical products were identified. Although most of these literature results could be used in our research to explain the mechanisms of photolysis-electrochemical detection of the original amino acids and peptides, separate studies were carried out to identify the photoproducts of certain amino acids, peptides, or proteins in ou...

Column Liquid Chromatography: Equipment and Instrumentation

“…The simultaneous measurement of monamines, metabolites, and amino acids in brain tissue and microdialysis perfusates was developed by Gamache and co-workers (F38), using HPLC with a coulometric array of graphite flow-through electrodes. Electrochemical detection of peptides and proteins was achieved with a postcolumn photochemical derivatization step, as reported by Krull and Dou (F39,F40), leading to the formation of electrochemically oxidizable photoproducts. Amperometric and fast-scan-rate cyclic voltammetry at a 10-jum-diameter platinum microelectrode were utilized by Soucaze-Guillous and co-workers (F41) for the detection and in situ identification of fullerenes which were separated by gel permeation HPLC.…”

Electrochemical detection as an alternative to UV in RP‐HPLC peptide mapping

“…It has been shown that some polypeptides can be detected by their free amino (NH,) group [38] or by the presence of the phenol group of tyrosine (tyr) or the indole group of tryptophan (trp) [39], methionine (met), and cysteine (cys). The use of photolytic reactions [35][36][37]431 and chemically modified electrodes [44] has extended the application of electrochemical detection [45]. Initial stuches showed that peptides could be detected by modifying them with electroactive substituents such as 3,G-dinitrophthalic anhydride [ 461.…”