Automated Solid Phase-Sequencing Using Fluorescent Edman Reagents

Abstract

Most commonly, in gas, liquid and solid phase sequencers the classical Edman reagent phenyl isothiocyanate is used, consequently the detection techniques of the released amino acid derivatives exploit the UV absorbent properties of the aromatic ring system. If high detection sensitivity is required, however, certain limitations exist given by the molar absorption coefficient of the chromophore as well as by the appearance of background contaminants of more or less known structure which are caused by side reactions of the degradation. Introduction of the intensely red azo chromo­phore in form of the DABITC (4-N,N-dimethylaminoazobenzene 4’-isothiocyanate) reagent in the manual and automated solid phase technology [1–3] with a concomitant shift of the detection wavelength into the visible range yielded a perceptibly cleaner microsequencing pattern. Fluorescence detection, although in general a magnitude of order more sensitive than colorimetry, has so far been largely unex­plored for sequencing purposes. Fluorescein isothiocyanate has been advocated for microsequencing [4], however, chemi­cally reactive side chains of the fluorophor like carboxyl and hydroxyl functions lead to the formation of additional byproducts making unambiguous interpretation difficult (Dr. Jin, personal communication, see also [5]). Recently, DNSAPITC (4-(N-1-dimethylaminonaphthalene-5-sulfonylamino) phenyl isothiocyanate as an Edman-type reagent carrying the dansyl fluorophor as a side chain has been proposed [6]. In this study the suitability of this compound and other ali­phatic fluorescent isothiocyanate homologues is tested for microsequencing with emphasis given to automated solid phase sequencing.