Reflections of a Fortunate Biochemist

Abstract

While contemplating the writing of this article I reread some of my early publications and in so doing was appalled at my profound ignorance. That, of course, is the advantage of hindsight. It is also an indication of how much has been learned by me and by others during the intervening half-century. That is the beauty of science; it is a collaborative work in progress that builds knowledge and understanding of the real world. I hope it may be interesting (for readers concerned with the process as well as with the results) to recount how we progressed from abysmal naiveté to our current informed view of the biology of oxidation stress. The goal of modesty is served by allowing for the probability that our successors 5 decades hence will consider the knowledge we have achieved pitifully incomplete. Sulfite OxidationAbraham Mazur introduced me to the wonders of biochemistry in an undergraduate course and then during a year spent working in his laboratory at Cornell Medical School. At the end of that year he recommended graduate work and sent me to Duke Medical School to work with Philip Handler, the chairman of the Department of Biochemistry. Handler was the most impressive person I have ever met. He was blessed with a photographic memory, an incomparable mastery of language, and excellent judgment. He assigned me to work with Murray Heimberg, who was then a senior graduate student. He was older than the usual graduate student, having devoted several years to fighting World War II. At one time during this conflict Murray had lost half of his body weight because of malnutrition while a prisoner of war, but he had entirely recovered when I knew him.We worked long hours, measuring O 2 uptake with Warburg microrespirometers and methylene blue bleaching in evacuated Thunberg tubes with a Coleman colorimeter. We found that ␣-hydroxysulfonic acids dissociated to carbonyl compounds plus sulfite and that sulfite was then oxidized to sulfate in the liver extracts we were studying (1). Thus began my long infatuation with sulfite oxidation.It had already been established that sulfite was readily oxidized by a free radical chain mechanism, but I did not know this and had to discover it for myself. Thus I found that sulfite could reduce cytochrome c and, of course, at the same time cytochrome c could oxidize sulfite. When this was done anaerobically the stoichiometry was 2 cytochrome c reduced per sulfite oxidized, as expected. However, in the presence of dissolved O 2 thousands of sulfites were oxidized per cytochrome c reduced. This could be rationalized by assuming that each time sulfite transferred an electron to cytochrome c, the resulting sulfur trioxyradical started a chain reaction between sulfite and O 2 as follows.

Distinct hyperpolarizing and relaxant roles for gap junctions and endothelium-derived H 2 O 2 in NO-independent relaxations of rabbit arteries

“…Notably, subintimal hyperpolarizations to A23187 and ACh were unaffected by catalase, indicating that endothelium-derived H 2 O 2 cannot be considered as a hyperpolarizing factor. Confirmation that exogenous H 2 O 2 can mimic the response to A23187 was obtained in endotheliumdenuded arteries exposed to X͞XO, which generates H 2 O 2 directly and via dismutation of the superoxide anions also produced by this system (22). X͞XO thus evoked catalasesensitive endothelium-independent relaxations that were maximally equivalent to Ϸ80% of induced tone but were associated with smooth muscle hyperpolarizations that were Ͻ5 mV, and therefore much smaller than the electrical response to A23187 and ACh.…”

Kinetics studies of the superoxide-mediated electron transfer reactions between rubredoxin-type proteins and superoxide reductases

“…Moreover, for all experiments, 150 U ml À1 bovine catalase was added to the reaction mixture, in order to remove H 2 O 2 , which is not only a product of the SOR reaction, but is also a by-product of the xanthine/xanthine oxidase superoxide-generating system [52,53].…”

Low intracellular zinc induces oxidative DNA damage, disrupts p53, NFκB, and AP1 DNA binding, and affects DNA repair in a rat glioma cell line

“…Briefly, cells were lysed in 0.1% Triton X and under two subsequent freeze͞rethaw cycles and stored on ice. SOD activity was determined from the percentage inhibition of the cytochrome c, xanthine-xanthine oxidase assay (29). The reduction of cytochrome c by superoxide generated from xanthine and xanthine oxidase is monitored by absorption at 418 nm.…”

“…CuZnSOD and manganese SOD activity was determined as described by Fridovich (29). Briefly, cells were lysed in 0.1% Triton X and under two subsequent freeze͞rethaw cycles and stored on ice.…”