00:00:00 You are about to see an interview with Dr. Ronald Estabrook, conducted by Professor Francis

00:00:29 Scott of the Department of Chemistry at the Rochester Institute of Technology.

00:00:35 Ronald Estabrook received his B.S. in biology in 1950 at Rensselaer Polytechnic Institute.

00:00:42 He then did a doctorate in biochemistry at the University of Rochester in Rochester,

00:00:46 New York, working in the general area of mitochondrial function under Dr. Elmer Stotz.

00:00:53 Dr. Estabrook's thesis dealt with studies on the cytochromes of heart muscle extract.

00:00:58 He moved as a postdoctoral fellow to the University of Pennsylvania School of Medicine, and there

00:01:04 he worked under the direction of Dr. Britton Chance.

00:01:07 Dr. Estabrook says he was very fortunate to get a visiting research fellowship to go to

00:01:12 Cambridge University in England for the year 1958-59, where he worked for Dr. David Kylan,

00:01:19 one of the pioneers in cytochrome biochemistry.

00:01:22 Dr. Estabrook returned to Philadelphia in 1959 and spent the next nine years there as

00:01:28 professor of physical biochemistry in the Department of Biophysics and Physical Biochemistry

00:01:33 in the School of Medicine of the University of Pennsylvania.

00:01:38 This was a particularly productive period in Estabrook's career.

00:01:42 During those years, he demonstrated the role of cytochrome P450 in many metabolic reactions.

00:01:48 His work attracted worldwide attention and brought many visitors to his laboratory.

00:01:54 In 1968, he moved to his present position as professor of biochemistry and chairman

00:02:00 of the Department of Biochemistry at the University of Texas Health Science Center in Dallas.

00:02:07 Shortly after his arrival, he was awarded a personal chair in biochemistry called the

00:02:11 Virginia Lazenby O'Hara Professorship of Biochemistry.

00:02:17 His laboratory in Dallas over the past 14 years has been one of the world's foremost

00:02:21 in the area of cytochrome P450 structure, function, and chemistry.

00:02:30 Ron, the cytochrome P450 system has played a key role in the metabolism of foreign compounds,

00:02:41 xenobiotics, compounds that can cause cancer and so on.

00:02:46 And you have played a seminal role in the study of that system, particularly in Philadelphia

00:02:53 and, of course, then with your own fine research group back in Dallas.

00:02:57 Would you like to tell us a little bit about the Philadelphia story as far as cytochrome P450 is concerned?

00:03:04 Yes, that's a very interesting and, to me, a very rewarding period in my own scientific career

00:03:14 and one which I think can illustrate the many different turns that one can take

00:03:22 in terms of developing a program of research.

00:03:28 The period of 1950s, which is when this story began, was one of great excitement

00:03:37 in terms of understanding the mechanism of oxygen utilization in various tissues.

00:03:44 And primary emphasis was placed on mitochondria, in particular because the recognition of ATP synthesis

00:03:55 and all of the factors that influence this was still in its infancy, less than 10 years old.

00:04:03 There was, concomitant with the understanding of the mitochondrial electron transport system,

00:04:11 the beginnings of an understanding of what was called cyanide insensitive respiration.

00:04:18 It was known that the respiratory chain of the mitochondria was sensitive to inhibition by agents such as cyanide.

00:04:28 But there were, in some tissues, the ability of respiration to occur in the presence of cyanide.

00:04:36 It's interesting, as one reflects back, that during that period there were three different groups,

00:04:43 very active and, frankly, unrelated and not really communicating with one another.

00:04:51 Were they aware they were working on different aspects?

00:04:54 No, they were not, to my knowledge, aware at all. These three groups were the following.

00:04:58 There was a very active group at the Worcester Foundation concerned with the transformation of various steroids,

00:05:06 in particular the breakdown by the adrenal cortex of cholesterol to the various metabolically active steroid hormones.

00:05:15 And who was the leader in that?

00:05:17 The leader would be, Lou Engle was one, Oscar Hector was another,

00:05:22 Alex Zaffaroni was very intimately involved in that group.

00:05:26 There were a very powerful series of investigators. That was one group.

00:05:34 The second group was operative at the National Institute of what is now heart and lung and blood diseases.

00:05:43 This was a group headed by B.B. Brody, Steve Brody,

00:05:48 one of, again, the most powerful scientific groups but concerned, in this instance,

00:05:55 with toxicology and the degradative breakdown of various drugs.

00:06:01 Julie Axelrod was one of the participants in this group, Sid Udenfriend, Jim Gillette.

00:06:09 It was...

00:06:10 Were these largely biochemists? Was this a biochemical approach?

00:06:13 These were largely pharmacologists, interestingly.

00:06:16 So we have, at the Worcester Foundation in Massachusetts,

00:06:19 mainly endocrinologists concerned with steroid metabolism.

00:06:23 We have, at the National Institute of Health, mainly pharmacologists concerned with drug metabolism.

00:06:30 And a third group, which was at the University of Wisconsin, was Jim and Betty Miller principally,

00:06:36 concerned with the oxidative transformation and conversion of a number of compounds

00:06:42 that were then recognized to form cancer or to be cancer-causing, carcinogenic compounds.

00:06:50 And these were also pharmacologists rather than biochemists?

00:06:54 These were oncologists.

00:06:55 They were in the Department of Oncology at the McCardell Institute of Cancer Research, University of Wisconsin.

00:07:01 So here we have these three groups operative in the 50s.

00:07:05 All recognizing that there was associated with many cells, in particular the endoplasmic reticulum...

00:07:12 Of the liver.

00:07:13 Of the liver, of the adrenal, of steroidogenic organs,

00:07:19 an enzyme system that required oxygen for the oxidative transformation of the chemicals,

00:07:25 electrons in the form of reduced pyridine nucleotide,

00:07:29 and resulted in an oxidative degradation or alteration of the chemical system.

00:07:38 Now...

00:07:40 May I just come back in a second?

00:07:42 Yes, sir.

00:07:43 The steroid aspect of it, in a sense, would be benevolent towards the evolutionary sense.

00:07:47 It was the biosynthesis of metabolites that the body needed.

00:07:51 That's good.

00:07:52 The work at Wisconsin, it would be, in a sense, antagonistic with regard to the body.

00:07:56 That's good.

00:07:57 Because all the same enzyme systems were generating chemicals...

00:07:59 That's correct.

00:08:00 ...of potential hazard.

00:08:01 And, of course, the middle one, the drug ones, either are.

00:08:04 Can either be...

00:08:05 It's been drug toxicity or drug excretion.

00:08:08 That's correct.

00:08:10 Now, the other facet of this problem which took place were, again, were two more factors.

00:08:19 Howard Mason at the University of Oregon,

00:08:23 Osamu Hayashi in Japan,

00:08:26 were at that time doing studies with oxygen-18

00:08:30 and recognized that there were certain oxidative reactions which occurred,

00:08:35 called oxygenases,

00:08:37 which incorporated oxygen-18 into organic molecules.

00:08:41 These were called either mixed-function oxidation reactions,

00:08:46 mono-oxygenase reactions, or dioxygenase,

00:08:50 based upon whether one atom or two atoms of a molecule of oxygen,

00:08:55 atmospheric oxygen, were incorporated into the product.

00:09:00 So there were the chemical studies.

00:09:02 These were chemists that were doing O-18 studies.

00:09:06 Then there were the biochemists,

00:09:08 and in particular, the one that sticks most in my mind

00:09:15 were the group of which I was a part in Philadelphia,

00:09:19 working in Britton Chance's laboratory,

00:09:22 where we had the opportunity,

00:09:25 through the genius of Britt Chance

00:09:28 in the manner in which he developed instrumentation

00:09:32 that would permit the recording and monitoring

00:09:35 of spectral transition of hemoproteins

00:09:38 during the course of cellular oxidative reactions.

00:09:42 Now, his background, he was an electronics man in the Second World War?

00:09:46 He was an electronics man critical in the development of radars,

00:09:50 part of the MIT group,

00:09:52 interested in chemistry, in particular kinetics.

00:09:56 Still active, obviously.

00:09:58 Still very active, head of the Johnson Research Foundation,

00:10:01 University of Pennsylvania Medical School.

00:10:03 But because of his electronic background,

00:10:06 was able to develop instrumentation

00:10:08 which, using turbid particulate membrane fractions,

00:10:12 allowed one to record, through different spectrophotometry,

00:10:16 changes in optical spectrum

00:10:19 as a consequence of associated metabolic transformation.

00:10:23 Now, was that a quantum leap in design of UV instrumentation?

00:10:26 Absolutely.

00:10:27 It was UV instrumentation?

00:10:28 No, it was visible.

00:10:30 And the idea is, up to that time, these were too messy.

00:10:33 I mean, they were cloudy solutions.

00:10:36 The turbidity problem made it insurmountable.

00:10:39 And he solved that?

00:10:40 Yes.

00:10:41 By a method of different spectrophotometry.

00:10:44 And, of course, using his knowledge of the amplifier systems

00:10:49 that he gained from his work on radar,

00:10:51 and also the associated development of photosensitive cells,

00:10:57 which were able to detect small changes in light transmission.

00:11:04 Britt Chance, at that time, had with him some spectacular people.

00:11:10 He was truly a man that I consider the brightest man

00:11:16 that I've ever had the opportunity to meet or to work with.

00:11:20 Are we talking about, say, 54, 55, in terms of history?

00:11:23 This is about the mid-1950s.

00:11:26 There were, at that time, in the laboratory, working on mitochondria.

00:11:31 All of the work at that time was concerned with mitochondrial respiration.

00:11:35 A man named G.R. Williams, Ron Williams,

00:11:37 who is currently at the University of Toronto,

00:11:41 chairman of the Department of Biochemistry.

00:11:44 They were investigating what is known as metabolic regulation

00:11:50 or respiratory control of mitochondria during the associated formation of ATP.

00:11:56 And as part of Ron's interest, a man named A.N. Pappenheimer,

00:12:03 now a Harvard University faculty,

00:12:06 was working at that time on diphtheria toxin

00:12:10 and had the hypothesis that somehow this was related

00:12:14 to succinate dehydrogenase of the mitochondria.

00:12:17 In other words, the toxicity induced by the diphtheria toxin

00:12:20 was a metabolic toxicity.

00:12:22 Absolutely.

00:12:24 Chance, in his generosity,

00:12:28 we always had tremendous flow of individuals coming through the lab.

00:12:32 And Pappenheimer had contacted Chance,

00:12:36 came down and did a series of experiments

00:12:39 looking at the midgut of the cecropia, the silkworm of all sorts,

00:12:44 to see where diphtheria toxin might be inhibiting mitochondrial oxidative metabolism.

00:12:52 Was there any reason that he chose the silkworm for this?

00:12:55 I honestly don't know. I presume it was because of the sensitivity.

00:12:59 Now, Ron Williams was assigned the responsibility of working with Pappenheimer.

00:13:05 We each were given this job when visitors came to the lab

00:13:09 of working along with them and sort of helping them on the instrumentation.

00:13:13 Excuse me, Zachary. With regard to that aspect of it,

00:13:16 am I understanding clearly that one of the reasons,

00:13:19 apart from the bright people, is that the instrumentation at that time

00:13:22 available in that lab was unique in the world?

00:13:24 It was. It was the only source for this instrumentation.

00:13:27 So, in other words, people had to come. There was no commercial development of it.

00:13:31 Commercial development came 15 years later,

00:13:34 until the late 1960s before the first commercial instrumentation.

00:13:37 So if anybody wanted to do that kind of study with these particular kinds of solutions,

00:13:41 they came to Philadelphia and the Johnson Research Fund.

00:13:43 Right. Now, in the early 1960s,

00:13:47 there were a few labs around the world

00:13:51 which took up and followed Britt Chance's lead and developed these.

00:13:56 Martin Klingenberg in Munich, Bunji Hagihara in Osaka,

00:14:00 Lester Packer out in Berkeley, California.

00:14:04 But they were very limited.

00:14:06 And at the time you're talking about when Pappenheimer came, he came to the mountain.

00:14:09 He came to the mountain. The fountainhead. Absolutely.

00:14:12 And, as I say, there was a continual flow of people.

00:14:15 Britt himself couldn't take care of all of these.

00:14:18 So, as the young individuals, knowledgeable of the instruments,

00:14:22 we were assigned various people as they came through the lab.

00:14:25 Ron Williams was assigned to work with Pappenheimer.

00:14:29 Now, Ron was a very methodical and also a very intuitive chemist.

00:14:37 And, yes, he did the experiments he was supposed to do,

00:14:41 but he always used to do one or two more.

00:14:43 And one of them was part of our routine

00:14:47 in doing spectral analysis of turbid samples

00:14:50 was to look for unique carbon monoxide binding pigments.

00:14:54 And Ron, in the presence of a chemical-reductant sodium dithionite,

00:14:58 using this messy mid-gut preparation of the silkworm, Cecropia,

00:15:04 added carbon monoxide and, for the first time, to my knowledge,

00:15:09 saw the appearance of a very strong absorbance band

00:15:13 with a maximum at 450 nanometers in the blue part of the spectrum.

00:15:20 This, to my knowledge, was the first time

00:15:22 that anyone had ever seen this hemoprotein.

00:15:25 May I ask you, for those of us who are not familiar,

00:15:28 why did one normally use carbon monoxide atmospheres

00:15:32 to look for that inhibition?

00:15:34 Was it derived from Warburg?

00:15:36 How far back did that kind of biochemical technique go?

00:15:40 Oh, this goes back to the early days of Hoppe-Zeller, actually,

00:15:44 in terms of hemoglobin.

00:15:46 They used carbon monoxide as a ligand routinely in hemoprotein studies.

00:15:50 Absolutely.

00:15:51 There were three cyanide which react with a ferricene protein,

00:15:55 carbon monoxide react with a ferrous,

00:15:57 and the analogy of carbon monoxide and oxygen

00:16:00 made that always the interesting agent to look at.

00:16:03 These three simple ligands, cyanide, carbon monoxide, and oxygen,

00:16:07 biochemists were using as respiratory tools for a long time.

00:16:10 For a long time.

00:16:13 I didn't mean to interrupt you.

00:16:15 The carbon monoxide spectrum shows a nice band of 450.

00:16:18 Right, and they never published this, interestingly.

00:16:24 That remained, that was about 1954-55.

00:16:29 That remained sort of as a unique, unexplained observation,

00:16:34 which wasn't unusual,

00:16:36 because we were getting all sorts of preparations

00:16:39 that people would bring into the lab.

00:16:41 They bring in their little bottles, put it through your machines, get data,

00:16:44 and not quite know what to make of it.

00:16:46 That's correct, many times.

00:16:48 There are still pigments in bacteria,

00:16:51 carbon monoxide-binding pigments,

00:16:53 that could remain as lifetime work

00:16:57 for individuals that have not yet been fully identified or characterized

00:17:03 or pursued in any depth at all.

00:17:05 But was this information about that kind of pigment

00:17:07 shared with the other people in the lab?

00:17:09 Because it wasn't just Williams and Papanheimer

00:17:11 who knew about that unusual absorption band.

00:17:13 Did other people know about it as well, if they didn't publish it?

00:17:16 That's an interesting question.

00:17:21 The answer to it really centers again about Bert Chance.

00:17:27 He was a very dedicated, compulsive, and highly curious person.

00:17:34 And I can recall...

00:17:38 We used to laugh when you think about it.

00:17:41 We used to have what was almost a Pavlovian response,

00:17:45 because every noontime we would have a seminar.

00:17:49 And whenever a visitor came through,

00:17:51 he would discuss what he was doing, what he had found.

00:17:55 So we all knew, all the time.

00:17:58 We used to salivate whenever we'd go to a seminar,

00:18:00 because we were so accustomed to...

00:18:02 What was coming out of the machine, right?

00:18:04 That's right.

00:18:06 So we all knew about this.

00:18:08 It didn't ring a bell, however.

00:18:10 Well, because you said there were many others as well, anyway.

00:18:12 Keep on turning up. I'll keep on turning up.

00:18:14 Now, it was interesting.

00:18:16 During that time in the lab,

00:18:18 there was a Japanese Ryo Sato, S-A-T-O, from Osaka,

00:18:23 who had been with Hugo Terrell in Karolinska in Stockholm,

00:18:29 who was working at that time, actually with me,

00:18:33 on a cytochrome from a salt-tolerant bacteria,

00:18:37 Helobacter.

00:18:40 Interesting.

00:18:41 A human protein which also still remains to be further characterized.

00:18:46 And Ryo was a smart man.

00:18:50 He was interested in mammalian cytochromes and unique cytochromes,

00:18:54 and was aware of the cytochrome P450, as it is now called.

00:18:58 And, in fact, Sato named it cytochrome P450.

00:19:02 The P450 meaning that the absorbance band

00:19:06 of the carbon monoxide ligand of the reduced hemo protein

00:19:10 was in the blue part of the spectrum of 450 nanometers.

00:19:14 Now, Sato did not work on P450 in Philadelphia.

00:19:20 He went back to Osaka, had a student, Suneo Omura,

00:19:25 and as part of this student's dissertation,

00:19:28 he was to work on the CO binding pigment.

00:19:31 And this culminated in the early 1960s

00:19:35 in two papers in the Journal of Biological Chemistry,

00:19:39 which were the first chemical definition

00:19:42 that a cytochrome P450 was a hemoprotein,

00:19:46 had protoheme as its prosthetic group,

00:19:49 was very labile in terms of its sensitivity

00:19:54 to detergents, to agents that would perturb the membrane.

00:20:01 In other words, when anybody tried to isolate it,

00:20:03 they messed it up.

00:20:04 Absolutely.

00:20:05 Made it very difficult to study.

00:20:09 It was interesting that at about the mid-1950s,

00:20:14 when Ron Williams, working with Papenheimer,

00:20:18 made this initial observation,

00:20:21 Martin Klingenberg was a visiting post-doctorate fellow from Germany.

00:20:28 Klingenberg was looking for a problem,

00:20:31 and he was working on liver microsomes,

00:20:37 and part of his spectral analysis of liver microsomes

00:20:40 recognized that cytochrome P450 was an integral part.

00:20:45 Excuse me now.

00:20:46 The other man had been doing it in the silkworm.

00:20:48 This man was doing it in rat microsomes.

00:20:51 That's correct.

00:20:52 Because in the manner of preparation,

00:20:54 now you have to remember,

00:20:55 the major emphasis in the lab was mitochondria.

00:20:57 One would take the liver of an animal,

00:21:00 homogenize it,

00:21:01 subject it to differential centrifugation,

00:21:04 isolate the mitochondria,

00:21:06 and the remaining supernatant contained the microsomal fraction

00:21:10 from the endoplasmic reticulum plus the soluble proteins.

00:21:14 So that was always left over.

00:21:16 Klingenberg took what was left over after the mitochondria,

00:21:20 subjected that to higher studies.

00:21:22 Same kind of study.

00:21:23 Right.

00:21:24 In order to get this bright red pellet of microsomes,

00:21:27 which we know contains cytochrome P450 today.

00:21:31 He did some very, very interesting studies.

00:21:34 He was more interested in the associated heme protein,

00:21:38 cytochrome B5.

00:21:40 It's another whole story that's in microsomes.

00:21:43 But he did some of the classic work

00:21:46 in identification of cytochrome P450.

00:21:50 So he had to go back to Germany.

00:21:52 He had actually been sent by Ted Bucher

00:21:56 in order to learn Chance's instrumentation.

00:21:58 Bucher was concerned with the characterization

00:22:03 of flight muscle mitochondria

00:22:06 and some of the factors that influenced

00:22:09 the oxidation of glycerophosphate in agents like this.

00:22:13 Now, Britain Chance didn't have any reservation

00:22:15 about people coming in saying,

00:22:16 I want to look at your black box to be able to take it off you.

00:22:19 He was not that kind of scientist.

00:22:20 There were so many problems to do.

00:22:22 Right.

00:22:23 There was no reservation feeling about it.

00:22:25 No paranoia about this at all.

00:22:27 There was a tremendous sharing of information and knowledge,

00:22:31 which really made it an exciting place

00:22:34 because everybody knew what everybody else was doing.

00:22:38 It really was one of biochemistry's little nuclei of development.

00:22:41 It was exciting.

00:22:44 But anyhow, Klingenberg wrote up his information.

00:22:47 I remember the package very well.

00:22:49 It was a brown envelope with all of this data

00:22:52 in the pseudo-English writing.

00:22:56 And the question came, you know, what to do with it.

00:22:59 Now, none of us had the time or the energy or the interest

00:23:02 to sit down and rework this information.

00:23:07 And David Garfinkel came from John Edsel's lab.

00:23:11 He was a Raman resonance spectroscopist.

00:23:13 And John Edsel was at Penn.

00:23:15 No, John Edsel was at Harvard,

00:23:17 one of the pioneers of biochemistry.

00:23:21 And he had persuaded Britain Chance

00:23:24 that David Garfinkel would be a good addition to the lab.

00:23:29 He was a spectroscopist.

00:23:31 And his responsibility was to rewrite the Klingenberg papers.

00:23:38 And this he did, and they appeared in archives of biochemistry about 1958.

00:23:43 But he himself became very interested

00:23:45 and did a study of the biological distribution of cytochrome P450.

00:23:50 Mind you, all of this was at a time before this pigment was even named.

00:23:54 It was just a carbon monoxide binding.

00:23:57 It wasn't even known to be a hemoprotein.

00:24:00 It was a carbon monoxide binding pigment

00:24:03 with an absorbance maximum of 450 nanometers.

00:24:06 It wasn't until 1961, when Sato and Omura recognized it to be a hemoprotein,

00:24:12 that the term cytochrome was applied.

00:24:16 Now, that was really the background.

00:24:20 And this is the explanation why, when I looked back at the study,

00:24:24 that there were these two papers by two separate individuals

00:24:27 in that journal, archives of biochemistry and biophysics.

00:24:30 Martin Klingenberg and David Garfinkel.

00:24:33 On their own, and that essentially ended their studies.

00:24:37 Martin went back to Munich, and David went on to his computer studies.

00:24:40 That's correct.

00:24:42 Martin Klingenberg, of course, has devoted the last 15, 20 years

00:24:47 to understanding oxidative phosphorylation,

00:24:50 in particular the adenine nucleotide translocases in the mitochondria.

00:24:55 David Garfinkel moved into the computer business,

00:24:58 computer simulation of metabolic sequences,

00:25:02 in particular those concerned with the Pasteur effect.

00:25:05 I've just read two papers of his on the modeling of the ischemic heart.

00:25:09 Absolutely.

00:25:10 Very nice.

00:25:11 Very, very brilliant work.

00:25:13 But they just had, you know...

00:25:15 They came and went.

00:25:17 As post-doctorate fellows, they were given a problem.

00:25:21 My own involvement was more on the side during this period.

00:25:28 I was aware of what was going on.

00:25:30 And your Ph.D. had been in respiratory, in mitochondrial...

00:25:33 Mitochondrial cytochromes here at the University of Rochester with Elmer Stotz.

00:25:37 And I was concerned really with the intricacies of what was called

00:25:43 the cytochrome BC1C part of the mitochondrial...

00:25:47 Complex, right.

00:25:49 Where antamycin was the inhibitor.

00:25:51 And I was working on that computer simulation and so forth.

00:25:56 At Pennsylvania, again, it's the spirit of the times,

00:26:00 there was an organization called the John Morgan Society.

00:26:05 John Morgan was the founder of the University of Pennsylvania Medical School

00:26:10 in the late 1700s.

00:26:16 Their honorary medical society was to bring together clinicians

00:26:21 and basic scientists on the staff of the University of Pennsylvania Medical School.

00:26:27 Just as a dining club, you said?

00:26:29 Just as a dining club.

00:26:30 We used to meet four times a year.

00:26:32 And four or five people would be designated

00:26:34 to get up and talk about what they were doing.

00:26:36 And it was at one of these meetings I met David Cooper.

00:26:40 David Cooper was a board-certified surgeon

00:26:43 in the Harrison Department of Surgical Research

00:26:46 who was very concerned with hypertension and steroid transformation.

00:26:53 He was firmly convinced, as it was understood in that time,

00:26:57 that steroids from the adrenal were somehow critically involved in hypertension.

00:27:03 And David, in his reading, had read a paper of Ryan and Engel,

00:27:08 they were at Harvard,

00:27:10 about the oxidative conversion of steroids,

00:27:14 in particular the 21-hydroxylation, the 11-beta-hydroxylation.

00:27:18 And very simplistically his concept was

00:27:20 that biosynthesis of steroids might be involved

00:27:23 in the evolution of hypertension in certain individuals.

00:27:26 Absolutely.

00:27:27 In the autology, right.

00:27:29 And so he became concerned and interested in knowing

00:27:33 how are steroids interconverted?

00:27:35 Now, you remember I told you that, oh, perhaps half a dozen years earlier,

00:27:40 the Worcester Foundation had this powerful group

00:27:43 looking at steroid interconversions.

00:27:46 And we went back to that literature,

00:27:48 and in particular the key paper was one by Ryan and Engel

00:27:51 in the Journal of Biological Chemistry,

00:27:54 who recognized that the oxidative transformation of some steroids

00:28:02 was sensitive to inhibition by carbon monoxide.

00:28:05 Well, I'd worked with mitochondria,

00:28:07 and I had been brought up more or less in the lore of the Warburg School,

00:28:12 which Chance was very knowledgeable of.

00:28:16 And Dave Cooper approached me about this problem and said,

00:28:23 how does one measure carbon monoxide inhibition

00:28:26 of an oxygen-requiring reaction?

00:28:29 Because he was interested in the chemistry,

00:28:31 and you had the access to the instrumentation to look at the mechanism.

00:28:34 Absolutely.

00:28:35 So we came together, and we started to look and see

00:28:39 what was the mechanism of carbon monoxide inhibition of steroid transformation,

00:28:43 in particular the 21-hydroxylation of progesterone

00:28:48 by the microsomal fraction of the B-fadrenal cortex.

00:28:54 So he used adrenals because it was steroid chemistry, right?

00:28:58 Absolutely. The reaction was very fast.

00:29:00 The assay, the Zimmermann assay, was very simple

00:29:04 to look at the 21-hydroxy derivative.

00:29:09 We went back, and we repeated the experiments of Ryan and Engel,

00:29:12 and indeed they were right.

00:29:14 The reaction was carbon monoxide inhibited.

00:29:17 It was...

00:29:18 Now, when you say inhibited, do you mean it stopped?

00:29:21 All the products were formed?

00:29:22 No, it depended on the ratio of carbon monoxide to oxygen.

00:29:26 The competitive inhibition of carbon monoxide to oxygen...

00:29:29 And this was a kinetic inhibition?

00:29:30 It was a kinetic inhibition.

00:29:33 I was aware of the work of Otto Warburg,

00:29:38 and we then thought if the reaction is carbon monoxide inhibited,

00:29:44 perhaps it's light-reversible.

00:29:47 And so we set up the apparatus to photodissociate the system

00:29:53 and measure its photochemical action spectrum.

00:29:56 Key to this whole arrangement at that time was a man named Otto Rosenthal.

00:30:02 Otto Rosenthal was a German biochemist who had been in Berlin,

00:30:08 knowledgeable also of the Warburg technique,

00:30:12 was working very close with David Cooper in the Harrison Department of Surgery,

00:30:19 and he was just a wealth of information.

00:30:23 In other words, he had the prior technique of Warburg,

00:30:26 and you had the chance...

00:30:28 I could look at the turbid samples.

00:30:31 He had the Warburg apparatus in terms of measuring oxygen utilization,

00:30:37 the ability to poise the system with various carbon monoxide-oxygen ratios.

00:30:42 All that we needed were a very powerful light source

00:30:47 and a source of monochromatic filters.

00:30:50 And that presented a problem.

00:30:52 We spent perhaps, oh, a couple of months

00:30:57 attempting to use a Bausch and Lomb grating monochromator

00:31:01 as the source of light for photodissociation.

00:31:05 Would the principle be that you were going to quench the reaction with carbon monoxide

00:31:09 and build up some key species

00:31:12 and then pop the carbon monoxide off under light stimulation?

00:31:15 That's correct.

00:31:16 The principle of the Warburg photochemical action spectrum technique

00:31:20 is that the maximum reversal of carbon monoxide inhibition by light

00:31:26 occurred at the wavelength where maximum absorbance

00:31:30 of the carbon monoxide derivative occurred.

00:31:33 This is what Warburg had used to identify cytochrome oxidase of mitochondria

00:31:38 as the terminal oxygen-interacting species.

00:31:41 Now, we tried these abortive experiments,

00:31:46 and it was apparent that we simply couldn't get enough light into the system.

00:31:51 We then went out, and I can remember the great difficulties

00:31:59 in getting the money to get a xenon lamp

00:32:03 that would permit us to get enough light energy

00:32:07 in the presence of monochromatic filters

00:32:09 to do some photochemical action spectrum.

00:32:13 We used an old Warburg apparatus

00:32:16 that Otto Rosenthal had brought with him from Germany, from Berlin.

00:32:19 It was the second or third Warburg apparatus in the world.

00:32:23 It was so ancient.

00:32:25 We borrowed monochromatic filters from a man named Lionel Jaffe.

00:32:30 I remember him very well, who was in the biology department,

00:32:33 who was at that time looking at photosynthetic reactions

00:32:38 and had the filters in order to do the photo action spectrum for photosynthesis.

00:32:44 Then we got the xenon lamp using first a light microscope lamp,

00:32:49 but that didn't give us sufficient energy either

00:32:52 to really do the experiments well.

00:32:54 And sitting and doing these experiments

00:32:58 and seeing for the first time

00:33:01 that irradiation of the carbon monoxide inhibited system

00:33:05 with light of 450 nanometers caused maximal reversal.

00:33:10 Now you didn't obviously know this before you started,

00:33:13 and what you did was you irradiated a series of wavelengths

00:33:16 to look at the efficiency of reversal.

00:33:18 Absolutely.

00:33:19 And lo and behold, smack in there was 450 responding.

00:33:21 Absolutely.

00:33:23 With my previous knowledge of the 450 work

00:33:29 that Klingenberg and Sato and G.R. Williams and Pappenheimer

00:33:35 and Garfinkel had done,

00:33:37 when we saw maximal reversal was at 450 nanometers,

00:33:41 the first thing we did was to go over

00:33:43 and put these microsomes in a spectrophotometer,

00:33:46 and lo and behold, it was loaded with P450.

00:33:50 And so that was the first realization of the function of the sensor.

00:33:55 That's right.

00:33:56 Now, that's a historic paper.

00:33:59 I mean, it changed all your lives more or less.

00:34:01 It certainly changed yours.

00:34:03 As luck would have it,

00:34:05 there was a further piece to the puzzle.

00:34:09 And, you know, science works on the ability

00:34:14 to make a sort of serendipitous observation,

00:34:19 but also you've got to be able to communicate this in a timely fashion.

00:34:24 I, through working in Chance's lab, had gotten to know

00:34:28 many of the leading investigators in Germany.

00:34:32 One in particular was Benno Hess,

00:34:34 who is now director of the Max Planck Institute in Dortmund.

00:34:38 Benno Hess was, at that time in Heidelberg,

00:34:42 was editor of Biochemica Zeitschrift

00:34:46 and was given the responsibility with Theodor Lehman

00:34:50 of developing a festschrift for Otto Warburg's 70th birthday.

00:34:56 This would be 60, 61.

00:34:57 About 60 or 61.

00:34:59 And I was asked by Benno, would I like to contribute?

00:35:05 This was just at the moment that all these experiments were breaking.

00:35:12 He said, yes.

00:35:13 We worked very hard on a very close schedule to meet the deadline.

00:35:17 And that paper was out in very short order,

00:35:19 and I think a very important paper, if I say so myself.

00:35:22 Now, this was the German, though, not your publication in English.

00:35:26 No, it was in English.

00:35:27 Where did it appear?

00:35:29 It was in Biochemica Zeitschrift, in the Warburg festschrift issue.

00:35:34 That was interesting because, really,

00:35:36 we had a great deal of trouble in those times

00:35:39 getting the information published.

00:35:42 You mean biochemistry wasn't popular?

00:35:45 Was there editorial difficulty?

00:35:47 Well, everybody believed that the major oxygen-utilizing reaction

00:35:52 within the cell was mitochondria.

00:35:54 Not these queer pigments.

00:35:55 Not these queer pigments and not these minor reactions.

00:35:59 Reactions which, in retrospect, we know

00:36:02 are critically involved in the problem of chemical carcinogenesis

00:36:07 and drug metabolism, steroid metabolism and all.

00:36:10 But in terms of respiratory, they were fringe at that time.

00:36:13 That's correct.

00:36:15 Now, again, Dave Cooper, in his manner,

00:36:21 which it's hard for me to explain,

00:36:24 he had an intuition as to where things were.

00:36:28 As soon as we recognized the key role of P450 in the adrenal system...

00:36:34 And it was still not called P450?

00:36:36 It was the carbon monoxide binding pigment of microstone.

00:36:43 He put together the work, and from his knowledge of pharmacology,

00:36:47 that there was a comparable enzyme system in the liver

00:36:51 which was responsible for the metabolism

00:36:54 and oxidative breakdown of many drugs.

00:36:56 Now, that was Brody's group.

00:36:58 That was Brody's group.

00:36:59 So we immediately set up the assay

00:37:01 to look at the conversion of codeine to morphine,

00:37:04 an oxidative demethylation reaction.

00:37:07 Formaldehyde was easy to measure,

00:37:09 and so we started out looking at that system.

00:37:14 Now, was your technique the same in the sense

00:37:16 it was inhibition of the key reaction mechanistically by carbon monoxide...

00:37:20 Carbon monoxide.

00:37:21 ...and popping it off at a series of wavelengths

00:37:23 and seeing whether the wavelength that it popped off

00:37:25 had the maximum at the P450?

00:37:27 That's correct.

00:37:28 We did this with the codeine to morphine system in liver.

00:37:33 We did it with acid-aniline hydroxylation.

00:37:36 In other words, all the stuff that Brody had published in the 50s...

00:37:38 Absolutely.

00:37:39 ...you looked at it again.

00:37:40 Absolutely.

00:37:41 We looked at the analgesic aminopyrine,

00:37:45 which is also undergoing demethylation,

00:37:48 and every one of them had the same pattern,

00:37:51 that the maximal reversal of carbon monoxide inhibition

00:37:55 occurred when we irradiated the sample with 450 nanometer light.

00:38:01 It's now been that methodology.

00:38:03 We stopped, very frankly, at that point,

00:38:06 because you could go through the whole Merck index

00:38:11 and look at all sorts of compounds,

00:38:14 and many, many more have been looked at by other investigators.

00:38:17 Historically, that would be 62, 63, 64.

00:38:20 That's correct.

00:38:21 There was Estabrook, Cooper, and Rosenthal in Philadelphia,

00:38:24 and Omura and Sata in Japan had put together suddenly

00:38:27 and said to the rest of the endocrinologists

00:38:29 and the people in McArdle and the people at the NIH and said,

00:38:32 Look, we seem to have identified the pigment and the enzyme system

00:38:37 that's involved in all of your businesses,

00:38:39 in the steroid area, in the drug area, and in the carcinogenesis area.

00:38:43 And so 64 was the beginning, in a sense, of an explosion of interest.

00:38:48 Now, the key was a symposium held in 1965

00:38:51 at the Federation meetings in Atlantic City.

00:38:56 Now, you have to remember, and we must do justice to Howard Mason,

00:39:01 who had, even though this was unpopular,

00:39:05 had maintained a keen interest.

00:39:08 Now, he had identified, through electron spin resonance spectroscopy,

00:39:13 a pigment that he called FeX in the endoplasmic reticulum,

00:39:18 microsomes, which he felt was functional in these oxygen reactions.

00:39:24 And it was only about this time that FeX and cytochrome P450 became equated.

00:39:32 But Mason had maintained, but using an entirely different tact...

00:39:36 From the 50s, he had been hacking away...

00:39:38 A keen interest in this, right?

00:39:41 Sata, of course.

00:39:43 At this time, the group in Stockholm of Arrhenius and Ernster were just starting.

00:39:48 Now, had Arrhenius come to Philadelphia at that time?

00:39:52 Well, Arrhenius was a student of Ernster.

00:39:54 Ernster had come to Philadelphia.

00:39:56 Lars Ernster was a good personal friend.

00:39:58 How do you spell Ernster?

00:40:00 E-R-N-S-T-E-R. Lars Ernster.

00:40:03 And he had been through the Britten labs?

00:40:05 Been through the Britten-Chance lab.

00:40:07 In addition, however, had been through the George Pallotti labs in Rockefeller.

00:40:12 So he had the combination of the cell biology

00:40:15 plus the biophysical chemistry that he got in Chance's lab.

00:40:19 So Arrhenius, who had studied cell biology in Pallotti's lab,

00:40:23 was also interested in the endoplasmic reticulum

00:40:26 and had a student, Stenna Arrhenius,

00:40:29 who is professor of toxicological pathology

00:40:33 at the Karolinska Institute in Stockholm.

00:40:36 This was when he was a medical student,

00:40:38 looking at the endoplasmic reticulum in microcells.

00:40:42 They were, very frankly, right on our back in terms of drug metabolism.

00:40:48 Carbon monoxide inhibition.

00:40:49 As had Alan Connie when he was working in McCardell

00:40:52 in the chemical carcinogenesis.

00:40:54 But we were about the only lab that had the methodology

00:40:58 for the photochemical action spectrum.

00:41:01 Was it in 65 or 66 that Connie wrote the review

00:41:05 that talked about the inducing of these enzymes?

00:41:07 And that was in the steroid field.

00:41:09 No, that was in drug too.

00:41:12 See, again, the pieces, it's a rather complex jigsaw puzzle.

00:41:18 The pieces begin to fall in place.

00:41:20 There was a man named Herbert Remmer,

00:41:22 who was professor of toxicology at Tübingen.

00:41:25 And Herbert Remmer's main interest in the 50s

00:41:28 was what he called drug tolerance.

00:41:31 What is it that causes a tolerance to various drugs

00:41:35 on repetitive exposure of the animals to these?

00:41:38 And by that we mean that the drug essentially becomes less effective

00:41:41 because the same dose does not produce the same response.

00:41:44 Absolutely.

00:41:45 That's the way he was defining it.

00:41:46 That's right.

00:41:47 He was to give a lecture at the New York Academy of Sciences

00:41:51 about 1963, the Merker-Remmer paper.

00:41:56 He had read our paper in Biochemiker Zeitschrift,

00:42:00 followed by a paper we had written for Science magazine,

00:42:04 a brief article on drug metabolism,

00:42:06 which, by the way, we had great difficulty getting published.

00:42:10 It was a very, very difficult and persuasive time

00:42:18 to try and tell the editors that this was important.

00:42:20 It seems now extraordinary.

00:42:22 I know, but it was...

00:42:23 But maybe innovation always has its own entropy about getting accepted.

00:42:26 We had serious problems.

00:42:30 But Remmer was aware of this.

00:42:32 I remember he wrote to me and said he was going to be in New York

00:42:35 at the New York Academy of Sciences meeting.

00:42:37 What his background was, what his interests were.

00:42:39 He'd like to come and spend some time in the lab

00:42:42 to just learn about this pigment

00:42:44 and what its relationship might be.

00:42:46 He was an M.D. in background?

00:42:47 He was an M.D., pharmacologist, German,

00:42:50 a man of just boundless energy, absolutely.

00:42:54 Still active?

00:42:55 Still active.

00:42:56 60th birthday actually is about a month from now.

00:43:02 He came, and that was exciting also.

00:43:06 Because about that time,

00:43:09 I had been lecturing around the country

00:43:12 and was invited by the students to Syracuse University

00:43:16 at the medical center.

00:43:18 I met a young man named John Shankman

00:43:21 who was just finishing his degree in biochemistry.

00:43:24 And I persuaded John,

00:43:26 largely because of his ability to handle animals,

00:43:29 to come work with us.

00:43:31 I was a spectroscopist.

00:43:33 Dave Cooper was a steroid endocrinologist.

00:43:35 You needed a good pharmacologist.

00:43:37 We needed somebody that could handle animals.

00:43:40 And so John came,

00:43:42 and at about the same time as Herbert Remmer,

00:43:45 we started looking at phenobarbital effects

00:43:48 on the liver microsomal system.

00:43:50 Still looking now for this tolerance bit.

00:43:52 Absolutely.

00:43:53 And we saw that P450 was very responsive.

00:43:57 In a matter of three or four days,

00:43:59 one could triple the content of P450 in liver,

00:44:03 the CO-binding pigment,

00:44:05 as a consequence of treating animals with a barbiturate.

00:44:10 This was the answer to Remmer's whole hypothesis,

00:44:14 which he had predicted,

00:44:16 that there was an enzyme system which was built up,

00:44:19 which was responsible for drug tolerance.

00:44:21 In other words, it more efficiently scavenged the agent

00:44:24 so less of it was in the bloodstream

00:44:26 and therefore there was less effect.

00:44:28 Absolutely.

00:44:29 It caused a more rapid clearance of the drug

00:44:32 and transformation of it.

00:44:34 So that was exciting.

00:44:36 Now, it was also about this time, 1965,

00:44:41 that I was invited to what was called an ISOCS,

00:44:45 an International Symposium on Oxygenases and Oxidases,

00:44:50 organized by Howard Mason, Martin Morrison, and Sue King.

00:44:55 Sue King at Albany.

00:44:57 Dave and I, Dave Cooper and I,

00:44:59 we had some arguments about this, I'll remember,

00:45:02 because we were concerned, how was P450 functioning?

00:45:06 We knew what it was doing.

00:45:08 It was activating oxygen for insertion of that oxygen

00:45:11 into these organic molecules.

00:45:13 But how was it doing it?

00:45:15 Now, at that time, Mason had indicated,

00:45:17 his prior data had indicated,

00:45:19 that it was taking a single one of molecular oxygen's atoms

00:45:22 and making it incorporated,

00:45:25 was being reduced to water.

00:45:27 So that was the function that it was doing with the oxygen.

00:45:30 It was a mixed-function oxidase.

00:45:32 A mono-oxygenase in Haiti's terms.

00:45:34 That's correct.

00:45:36 And we were concerned, what was the mechanism?

00:45:40 And again, the way that the work went

00:45:45 was that we sort of worked from deadline to deadline

00:45:48 in terms of symposia.

00:45:49 Because there was tremendous worldwide interest.

00:45:51 Oh, absolutely.

00:45:52 Things were exploding all over the place.

00:45:54 It was really fantastic.

00:45:56 We had a lot of people passing through the lab.

00:45:59 A lot of communication going on

00:46:01 in terms of activities in various places around the world.

00:46:06 The question is, how was the P450 functioning?

00:46:09 And it was, I think, in that ISOC's paper,

00:46:12 which was about 15 years ago, about 1964-65,

00:46:18 and it was Dave Cooper who came up with this,

00:46:21 that the cyclic functioning of cytochrome P450

00:46:24 was first presented.

00:46:26 See, the question came, what was the sequence

00:46:29 of oxygen interaction, electron interaction,

00:46:32 substrate interaction?

00:46:34 In other words, you were aware that this enzyme,

00:46:36 or this system, had to bring the substrate

00:46:38 and the oxygen approximate enough together

00:46:41 that it could take one single atom

00:46:43 and put it in a particular bond in a particular position.

00:46:46 It wasn't random.

00:46:47 It was very selective.

00:46:48 But the question was, did the substrate

00:46:51 first bind to the enzyme...

00:46:53 And cause it to change.

00:46:55 And cause some sort of a change in the substrate

00:46:58 as a consequence of electron transport

00:47:00 from reduced pyridine nucleotide,

00:47:02 so the oxygen could then be inserted?

00:47:05 Or did the oxygen bind first and become activated,

00:47:10 followed then by the substrate?

00:47:12 And it was this sort of thinking which then led to this

00:47:15 now, I think, reasonably well-established cyclic function.

00:47:22 There remained a serious problem.

00:47:25 Sorry, with regard to your conclusions

00:47:27 that you put into that particular paper in 1965,

00:47:30 how did you come down one other side on this fence?

00:47:33 Did you decide the substrate went first?

00:47:36 I know you did.

00:47:37 We decided the substrate went first,

00:47:39 and this was really based on an observation

00:47:42 by a woman, Shak Narishimula,

00:47:46 who was working with Dave Cooper on the steroid system,

00:47:49 that observed what we now recognize

00:47:51 to be a spin-state transition

00:47:53 on substrate interaction with the ferric form of the heme protein.

00:47:57 In other words, when she added progesterone

00:48:01 to a clarified sample of adrenal microsomes,

00:48:05 she was trying to purify the system,

00:48:08 there was a spectral change.

00:48:11 Visible. I mean, there was a shift in the absorption.

00:48:14 Shift in the absorption spectrum.

00:48:16 At that time, Jim Gillette, Henry Sasamy,

00:48:19 Dave Cooper, Herbert Remmer, John Shankman,

00:48:22 were all working together in the lab,

00:48:26 and we said, all right, shock,

00:48:28 if that works on the adrenal system,

00:48:30 it ought to work on the liver system.

00:48:32 And lo and behold, it did.

00:48:34 And that's where the terminology now

00:48:36 of type 1 binding substrates and type 2 and so forth...

00:48:39 Irrespective of oxygen binding at all,

00:48:41 many ligands bound to the heme protein...

00:48:45 And caused a spin-state transition, that's right.

00:48:48 Well, that was the origin of the shift,

00:48:50 but it caused a thing you could measure in your ultraviolet machine.

00:48:53 Absolutely.

00:48:54 And that this followed the binding characteristics

00:48:57 of a substrate binding to an enzyme.

00:48:59 Saturable system,

00:49:01 dependent upon the concentration of the substrate,

00:49:04 an equilibrium process...

00:49:07 Sorry, for those who aren't knowledgeable in biophysics,

00:49:10 this wasn't a change in the redox state of the iron.

00:49:13 It wasn't a question of ferrous going to ferrous.

00:49:15 It was also a spin state.

00:49:17 A spin state, that's correct.

00:49:19 It was a transition from a low spin...

00:49:21 In other words, it was a rearrangement of the geometry

00:49:23 of the central metal atom and its ligands.

00:49:25 That's correct.

00:49:26 Putting it very simply.

00:49:27 That's correct.

00:49:28 So, we knew that substrate was binding first.

00:49:32 But there was a major problem that still existed,

00:49:35 and that was the cytochrome P450

00:49:38 appeared to be so fragile and so delicate

00:49:41 that every attempt to purify it

00:49:44 just simply went to naught.

00:49:49 Now, there was a man, Paul Talalay,

00:49:52 at Johns Hopkins who had a student,

00:49:54 and again, a man who just made one contribution

00:49:57 and has made no others in the area of P450,

00:50:01 was concerned with chemical carcinogenesis,

00:50:04 and in particular, polycyclic hydrocarbons

00:50:06 and how they were being metabolized.

00:50:09 And what Paul Talalay did, he had a student,

00:50:14 I honestly don't remember the student's name,

00:50:16 who attempted fractionation

00:50:18 in the presence of polyhydric alcohols, glycerol.

00:50:22 And this stabilized the membrane system enough

00:50:25 so it now could be purified.

00:50:28 And that has then led to the work that Kuhn has done.

00:50:32 Now, was the point of purification there

00:50:34 to permit much more accurate spectroscopic measurements

00:50:37 in terms of not just different spectra,

00:50:39 but absolute spectra, in other words, you're looking...

00:50:42 I'm sure.

00:50:43 Was that one of the incentives behind the purification drive,

00:50:47 or was it merely to learn about molecular weights

00:50:49 and peptide fractions and so on?

00:50:52 I think the original incentive

00:50:55 was the challenge to purify a membrane-bound protein.

00:51:00 Second was the ability to characterize

00:51:04 and measure the functionality

00:51:06 of this isolated purified heme protein

00:51:09 in a system that could be regulated

00:51:11 with regard to all the other perturbing factors.

00:51:15 I think third, although perhaps of a lower priority,

00:51:19 was that the understanding

00:51:22 of the mitochondrial respiratory chain

00:51:25 came as a consequence of having available inhibitors

00:51:29 that would allow you to dissect out

00:51:31 various portions of the respiratory chain.

00:51:34 We had no inhibitors other than carbon monoxide

00:51:37 for the microsomal hydroxylating system.

00:51:40 So you could prepare antibodies

00:51:42 once you had the purified material.

00:51:45 And I think also it was at a time

00:51:48 when understanding of membrane fluidity,

00:51:51 the role of lipids and the functionality of membranes,

00:51:54 all were of keen interest

00:51:56 to the biochemists and the biophysicists.

00:51:58 So that was part of the driving force.

00:52:00 It did lead, however,

00:52:02 to an unexpected and still unresolved problem,

00:52:05 and that is the multiplicities of cytochrome P450.

00:52:08 There are at least 6 to 8 different protein structure forms

00:52:15 of cytochrome P450

00:52:17 in just the endoplasmic reticulum of liver.

00:52:21 Within a single species, within a single animal,

00:52:25 the idea is that it's not just a question

00:52:28 of veering from rat to mouse to dog.

00:52:30 Within the rat, for example,

00:52:32 there's recognized now a multiplicity.

00:52:34 Absolutely.

00:52:35 Do you conceive that each one does a specific function?

00:52:39 That's what people are trying to determine.

00:52:41 In evolutionary terms, what would be the purpose of that?

00:52:44 One would look at specificity for substrate.

00:52:49 It has always been a problem with the P450 system

00:52:52 because it has such broad specificity

00:52:55 to do aromatic hydroxylations,

00:52:57 to do alkyl, aliphatic hydroxylations,

00:53:01 N-oxidations, O-dealkylation.

00:53:04 It's a fantastic little chemical lab.

00:53:06 Absolutely.

00:53:08 There are those that are looking for specificity,

00:53:12 and we still don't know the answer to this.

00:53:16 There appears to be some preference,

00:53:19 but no absolute specificity.

00:53:21 Are you saying, for example,

00:53:23 that amines might be metabolized by one set

00:53:27 or one particular kind of P450 alcohols by another?

00:53:30 That's correct.

00:53:31 That's the theory.

00:53:33 It isn't working quite that way

00:53:35 because even though they're able to isolate the heme protein,

00:53:40 it's extremely difficult to get it 100% pure.

00:53:44 You can get them 90% pure.

00:53:46 How could you tell?

00:53:47 How do they distinguish?

00:53:48 Chromatographically, the purity?

00:53:50 By polyacrylamide gel electrophoresis,

00:53:52 things of this nature.

00:53:53 A single point would be it.

00:53:55 And by N-group analysis of the amino acids,

00:53:58 things of this nature.

00:53:59 What about the antibody business?

00:54:00 Is the antibody selective enough

00:54:02 that they can say they'll pick off one?

00:54:04 This is a...

00:54:06 Depends who you believe.

00:54:08 All right?

00:54:09 It's an ongoing area.

00:54:10 It's an ongoing area.

00:54:12 Mechanism, of course, is the key issue.

00:54:15 How is oxygen activated

00:54:18 in order to be inserted

00:54:20 into all of these different organic molecules?

00:54:23 And I think for those who are not familiar

00:54:25 with this kind of chemistry,

00:54:27 this system does extraordinary things.

00:54:29 I mean, it'll take, for example, benzene

00:54:31 and metabolize it to phenol...

00:54:33 Absolutely.

00:54:34 ...which organic chemists find extremely difficult to do.

00:54:37 Or phenols and transform them to quinones.

00:54:40 Its activation is extremely unique

00:54:43 in terms of chemistry.

00:54:44 Absolutely.

00:54:45 And one of the most intriguing

00:54:50 and, I think, challenging aspects for the future

00:54:54 are the ability to isolate

00:54:57 the requisite oxygen-human-protein system,

00:55:02 immobilize it,

00:55:03 and then use this as a means of chemistry

00:55:07 to accomplish oxygen reactions

00:55:09 that the organic chemist now finds nearly impossible.

00:55:13 You're talking about as a synthetic tool.

00:55:15 As a synthetic tool.

00:55:16 You're not among things like an artificial liver.

00:55:19 Well, there are those that are considering at the moment

00:55:23 the use of incorporation of such a system

00:55:26 in dialysis in order to detoxify drugs that...

00:55:33 Dialysis won't pick up.

00:55:34 ...that dialysis won't pick up.

00:55:37 There are many, many practical applications.

00:55:40 Means of lowering blood cholesterol.

00:55:43 Crazy as it sounds.

00:55:45 You mean because it wouldn't metabolize it?

00:55:47 It would metabolize it,

00:55:48 but to products that could then be rapidly removed and excreted.

00:55:52 Now, may I...

00:55:53 Sorry, before we get into the kind of hard science here,

00:55:55 let's go back again.

00:55:56 You talked about 65, 66, 67

00:55:59 as an area where all this study was coming together.

00:56:02 Right.

00:56:03 Now, it was at that time then you went yourself to Dallas.

00:56:05 I went to Dallas in 68.

00:56:07 Yeah.

00:56:08 And you carried with you your interest in developing.

00:56:11 What aspect would you say...

00:56:12 But Chance was never really interested in the microsomal system.

00:56:14 He was a very generous man.

00:56:16 His main challenge was in mitochondria

00:56:19 and in cellular regulation of metabolism.

00:56:23 And he more or less handed me the microsomal system

00:56:28 and said, it's yours.

00:56:29 You have complete access to the lab,

00:56:31 all the instrumentation, all of his guidance...

00:56:33 This is why you're still there.

00:56:34 When I was there.

00:56:35 When I left, we sat and discussed.

00:56:37 And he said, Ron, he said,

00:56:39 microsomes and P450 is your business, you take it.

00:56:43 And with my blessing, and I'll help you.

00:56:45 Which was very generous because he had the opportunity

00:56:48 and I think the right as director of the lab

00:56:51 to say, you know, he wanted to keep that at the Johnson Foundation.

00:56:54 But he didn't.

00:56:56 And I moved to Dallas,

00:56:59 brought together what I think are some superb people.

00:57:03 Now what's the thrust?

00:57:05 I know you have many aspects,

00:57:07 but in your own mind, the principal contribution

00:57:09 that you're trying to do in that group,

00:57:11 is it mechanistic again?

00:57:13 Is it isolation?

00:57:14 Is it structure?

00:57:15 Are you still in the biophysics area?

00:57:17 I think all of those, to be frank with you.

00:57:19 But what would you feel...

00:57:21 What are we really trying to do?

00:57:24 Well, a single thrust, if you had to pick,

00:57:26 that what you would really like to solve is this aspect.

00:57:28 I don't want to give any information away to your competitors.

00:57:31 I have no concern about...

00:57:33 No, I think my own, in general terms, driving force

00:57:37 is to understand the system sufficiently

00:57:40 so that we have some mechanism of regulating and controlling.

00:57:47 I originally started out as a biophysicist, biochemist.

00:57:53 Over the years, I'm becoming more and more converted

00:57:56 to the potential importance of the system

00:58:00 in terms of understanding cellular toxicity and carcinogenicity.

00:58:04 Is the environmental toxicology area, for example,

00:58:07 is blossoming onto this insight, the toxicity aspect?

00:58:13 I was recently visiting a laboratory.

00:58:20 I won't say where.

00:58:22 Interested in polybrominated biphenyls.

00:58:26 Fire retardant, Fire Master II.

00:58:28 Infamous now.

00:58:29 Infamous now in Michigan.

00:58:31 And in the course of preparing for this,

00:58:34 I read that there were, in 1975,

00:58:36 literally millions of pounds of that compound prepared.

00:58:45 The genius of man in terms of his chemical abilities is fantastic,

00:58:52 yet we seem to live in a sea of chemistry

00:58:57 for which we have no true biological understanding.

00:59:01 No, because we have a chemical environment

00:59:04 in the sense we've synthesized many of these for useful purposes,

00:59:07 and now we're left with the recognition that they can be threats to us.

00:59:10 Absolutely.

00:59:11 Now, do you think many, in terms of our political setup,

00:59:14 do you think we overreact to that threat?

00:59:16 Yes. I don't think that the scientific data is all in.

00:59:20 And I think that, in particular, yes, there's a risk,

00:59:25 but I think somebody is going to have to come up

00:59:27 with a balance of risk versus benefit.