8: Spin Labels (Stanford)

The “spin label technique” involves probing the structures and dynamics of a biological system – usually a macromolecule or a membrane – with a paramagnetic molecule, usually a nitroxide free radical. Now widely applied, the technique has a background that can only be called adventitious. While still at CIT I was given a sample of a red liquid, a stable free radical, di-tert-butyl nitroxide. I think the sample came from DuPont via Professor Jack Roberts, but I am not certain of this. An experiment was carried out at CIT to see if this substance showed any ferromagnetism at low temperatures, with negative results. Somehow the sample followed me to Stanford, possibly because Hayes Griffith was interested in studying the resonance of paramagnetic inclusion compounds.


In addition to the above bit of history, I had been in correspondence for a year or more with a Professor Shun-ichi Ohnishi in Japan who wished to visit and work in my lab.  I had planned to give him a project involving triplet excitons in charge transfer complexes.  However when Ohnishi arrived in my office for the first time, he told me in no uncertain terms that he wanted to work in the field of biophysics.  By coincidence at about that time I was invited to a lunch in the medical school with Arthur Kornberg, Paul Berg, Manfred Eigen, and Josh Lederberg. There was an animated discussion of mutations produced by acridine dyes in bacteria and whether the dye molecules were isolated from one another when bound to DNA, or stacked one against the other.  It occurred to me that if the acridines were paramagnetic this could be easily resolved.  I also recalled that another three ring aromatic molecule, chlorpromazine was easily oxidized, and might mimic acridine in its interaction with DNA.


So I gave this problem to Ohnishi. The resonance spectra of the chlorpromazine cation did show that it bound to oriented DNA with its molecular plane perpendicular to the helix axis and certainly not stacked one cation against the other (98).


While these various things were going on, I had a number of conversations with Lubert Stryer concerning his use of fluorescent probes for biophysical studies. By this point it was obvious to try to use paramagnetic resonance with nitroxide radicals as probes, and I asked two postdocs, Stone and Buckman to try to prepare such nitroxide probes, or “spin labels” (106). They were successful, and Larry Berliner soon demonstrated their utility in binding to the active site of an enzyme (110). Hayes Griffith prepared a particularly useful labeling reagent, a nitroxide maleimide (107). Thereafter followed a rather large number of studies of labeled proteins, especially hemoglobin.


In my lab the most interesting spin labeling of proteins concerned hemoglobin. See list of publications for references. In recent years Wayne Hubbell has greatly advanced the usefulness of spin labels in the study of proteins through his site directed labeling methods.


In Addendum II, I have added some printed material that provides more detail concerning the earliest work with spin labels, taken with permission from the 2006 epr newsletter.



Chapter 9: Early History of Fluid Membranes