Protein Sequencing at the Picomole Level Using Color Ethnan Reagent

In 1972, I started my postgraduate studies in the lab of J.-L tin, at the National Taiwan Universit,~Taipei. The research project assigned to me was the synthesis of an analog of dansyl chloride (din%ethylaminonaphthalene sulfonyl chloride), which was both fluorescent and colored. Dansyl chloride was then a widely used fluorescent reagent for N-terminal analysis of proteins.’ To start the project, I was even 8 NT$ (30 cents US) to purchase a half kilogram of toilet deodorant (naphthalene ball) from a nearby store. The naplithalene was crystail’ized, nitrated, reduced, methylated, and then coupled to a dlazonlum salt The color appeared, but the fluorescence disappeared as a result of diazotization. Theexperiment was considered a faikire, and I was told to switch the project to study the carcinogenic effect of azodye on dsldcen embryos. I have always disliked doing experiments on animals. Determined to stay away from the chicken e~,I su~stedto tin that wo modify our original goal by synthesizing only the color version of dansyl chloride. We prepared dlmethylaminoezobenzene sulfonyl chloride (dabsyl chloride),2 a reagent that turned out to be very useful for amino addanalysis. Duringthe decade of the 19705, protein chemists began to encounter the pressing need for sensitive protein sequencing methods. The only practical method available then was Edinan (phenylisothiocyanate) degradation.3 As a result of my postgraduate woric, I conceived the Idea of synthesizing a color Edman reagent to improve the sensitivity of manual sequencing. Howover, I was only able to put thIs Idea Into practice two years later, when I undertook my PhD studies at the Australia, National University, Canberra. In the end, the synthesis and appilcatlon of a color .Eiknan reagent, dinethyl. aminoazobenzene Isothiocyanate (DABITC), was to become the major content of my PhD thes~ With DABIT~it became posnbk to monitor visually each step of the manual sequencIng. More imposlandy, as littleas 10 picomolesof the released N-terminal amino acids could be identified on a stanp-slzed thin-layer plate with the naked e~ I was genuinely delighted by these result, and believed that a method had beenhamd forprotein sequencing at the picomole level. I was eager to Ibid an application toprove it. In 1978, I brought the DA811C method to the lab of L Wittmann-Llebold at the Max Planch Institute in Berths. There, I relbied a protocol by adapting the DABITC/phenylisothlocyanate double coupling. The usefuhless of the DABITC technique was convincingly demonstrated as It foalilated the completion of the sequence analysis of nbosomal proteins. In my opinion, the popularity of the manual DABITC technique stems primarily from the ease and high sensitivity of analyzing the ccl. ored amino acid derivatives on thin-layer duomatography. To date, the DABITC manual sequencing method I, still used by many labs that cannot affoird to install an automatic sequencer. In our lab, the DABITC technique is routinely applied for one step quantitative N-terminal analysis (QNA)4 of pol~eptldes—.anextremely useful technique that was widely used In the 1960s and 1970s, but somehow got lost in the 1980s. CC/NUMBER 24 This Week’s Citation Classic ® 17.1991