In the essay, Arnold Martin Katz recalls his 1959 paper describing a modified method for the separation of peptides on filter paper. At the time, Katz was a research fellow at the National Heart Institute at NIH, in the lab of Christian Anfinsen, which focused on protein structure and function. (In 1972, Anfinsen shared the Nobel Prize in Chemistry with Stanford Moore and William Stein for his work showing that a protein's conformational structure is determined by its amino acid sequence.)
To separate peptides, researchers used 2D chromatography followed by electrophoresis. The chromatography was conducted in a large glass tank full of the solvent toluene, which was highly flammable; a single stray spark "would probably have taken two floors off the NIH building," Katz told The Scientist recently. Katz, William Dreyer and Anfinsen adapted the technique to use a non-explosive solvent instead. "We probably saved a few investigators' lives," he said. (Dreyer went on to invent an extremely sensitive method for protein and peptide sequencing in 1977.)
According to the Science Citation Index, the 1959 paper was cited 619 times between 1961 and 1975. Since then, it has been cited about 150 times. Though it was a nice technique at the time, Katz said, "science has moved on" to more sophisticated techniques using molecular biology and genetics.
Shortly after his two-year fellowship with Anfinsen, Katz moved on from studying protein structure and function to the biochemistry of muscle contractions, making several key contributions to understanding the role of calcium in cardiac function. He also became Chief of Cardiology at the University of Connecticut School of Medicine, a position he held for 25 years. He retired from both research and medical practice about ten years ago, though he continues to teach, write textbooks and research the history of medicine.
The paper: A.M. Katz, W.J. Dreyer, and C.B. Anfinsen, "Peptide separation by two-dimensional chromatography and electrophoresis," Journal of Biological Chemistry, 234:2897-900, 1959.
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I am somewhat amused to find this paper listed amongst the "most cited articles ever published" as this contribution is primarily methodological in scope, and the method wasn't even original to us! The value of this detailed description of a modification of the peptide "fingerprinting" method used earlier by V.M. Ingram probably lies primarily in our use of "varsol" in plastic, instead of toluene in glass, for cooling during the high voltage electrophoresis of 181/4 by 221/2 inch filter paper chromatograms. This modification was necessitated by the configuration of our NIH lab, where the hood (in which we did electrophoresis) was adjacent to the only exit. The thought of a spark --generated by the 2000 volt, 250 milliamp power supply -- igniting 10 gallons of toluene in a huge and fragile glass jar served as a most effective stimulus to this paper. Repeated demonstrations to visiting scientists (and girlfriends) of the explosive response of a few drops of toluene to a lighted match provided a vivid reminder of the hazards of the Ingram method. A number of us spent much time looking for other organic solvents, especially those which wouldn't permeate or dissolve plastics, which could be used in these peptide separations. As I recall, the use of varsol (a light petroleum fraction with a flash point well above room temperature) stemmed from several visits to the National Bureau of Standards and one to a local gas station.
The remainder of this method was relatively standard; the use of n-butanolacetic acid-water for paper chromatography was well established in Dr. Anfinsen's laboratory, and high voltage paper electrophoresis had been employed with considerable elegance by many, including Sanger's group. The combination of these principles, which separated peptides in two dimensions by methods based on different properties of each peptide, had, as indicated earlier, been used by Ingram to identify specific amino acid substitutions in abnormal human hemoglobins. By performing the chromatography first, unlike Ingram who did the electrophoresis first, we were able to improve the resolution of the "fingerprints" by taking advantage of the percolation of the buffer-applied to dried paper chromatograms -- towards the origin, which tended to 'sharpen' the bands.
At the onset, I indicated with some chagrin that this methodological paper, and not one of my more recent conceptual articles, has become a '"best seller". Yet history's judgment may not be wholly inappropriate. This paper was, in fact, the only full-length paper to have come from my two-year tenure as Research Associate in Dr. Anfinsen's laboratory at the NIH. I was, at that time, most jealous of my friends who were able to grind out large numbers of papers laden with data while I struggled for almost two years with methodology. In retrospect, however, I have come to appreciate the opportunity I had to learn many different techniques that proved invaluable in my later work. It is for this reason that the paper I wrote with Bill Dreyer and Chris Anfinsen not only calls up vivid memories of my scientific youth, but also serves as a reminder of the value of methodology in scientific observations.
Arnold Martin Katz
mail@the-scientist.com
Editor's note: this article has been updated from a previous version.
Links within this article:
A.M. Karz et al, "Peptide separation by two-dimensional chromatography and electrophoresis," Journal of Biological Chemistry, 234:2897-900, 1959.
http://www.the-scientist.com/pubmed/14404782
Christian Anfensen
http://nobelprize.org/
T. Sharrer, "The Dreyer peptide and protein sequencer," The Scientist, July 1, 2007.
http://www.the-scientist.com/article/display/53312/
HistCite citation data for the paper
http://garfield.library.upenn.edu/
