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ESR STUDIES OF BIOCHEMICAL EFFECTS OF ATMOSPHERIC POLLUTANTS TIM COUNCIL FOR TOBACCO RESEARCH, U. S. A. 633 Third Avenue New York, New York Attention: Dr. J. Morrison Brady Associated Scientific Director

lung into which had been smoked six popular brand cigasettes. A short pre- paramagnetic resonance signal from an.homogcnate of a freshly excised rabbit print of a publication, Appendix A, which outlines these results is included i with this proposal supplement. In our proposal 05-47Z7, I indicated that I had observed an electron In a telephone conversation I was asked if I could give some additional information to strengthen my grant application. cussion will supply the necessary information. I believe the following dis- As indicated in Appendix A, I believe the observed electron para- magnetic resonance pattern is produced by the interaction of free radicals and,(or ~.;.! . reactive diarnaonetic molecules with a metal complex present in the rabbit lungs. To be more specific I believe the bulk of the observed sional is due to the reaction of these free radicals and/or reactive molecules with the haer.zoglobin molecules present in the red blood cells. Such an interaction is represented In the haemoglobin molecule the iron is present as a ferric ionic ~,e3+. The paramagnetic resonance spectra of Fe3~ ionic complexcsA have been investigated by many workers. In particular however, detailed electron spin resonance studies have been performed on four derivatives of the haemoGlobin molecule, in order to discover the detailed form of bonding between the central iron atom and the particular group on the sixth coordination point, the point at which oxygen attachment occurs. 1.003545915 . The tcntativc: explanation of the reported ESR signal from smoked rabbit lung is the attachment of an active molecule (s) from tobacco smoke to the sel£sarne sixth coordination point. On the basis of this hypothesis several

Is this the transport mechanism to the liver which leads 0 the formation of acetonitrile by the liver? . Why does the lung membrane pass these molecules so readily? . What molecules or radicals are thus passed and picked up 'and are these identical to those in smoke? . Is thert a connection between these modified ha emoglobin molecules and the observed vessel constriction in smokers? Since none of these questions-lie within the scope of our present NCI program (primarily because of the unexpected nature of this discovery), TRC support there are several rather direct questions which must first be dealt with; for example: .attempt an exploration of the more complex biological questions raised above, s essential to allow the investigation of any or all of these. Before we . What types of molecules and/or radicals will react to form .haemoglobin complexes such as we have observed? 2. What is the stability of these complexes at physiological conditions ? 3. Are such complexes formed by other atmospheric contaminents;i. e. , smog, automobile exhaust fumes, etc. ?

THEORETICAL CONSIDERATIONS The structure of the central portion of the haemoglobin molecule is represented in Fig. 1 where the haem-porphyrin plane is shown perpen- dicular to the axis joining the iron atom to the globin molecule and the ligand is the relative ease with which the ligand of the sixth coordination point can be changed which makes it 'feasible to study a whole series of derivatives . at the sixth coordination point. The main feature of the hacmoglobin molecule formed by substituting different ligands into this position. Single crystal stud'ies have been made on the acid mathaemoglobin, acid metn-jyoglobin, metmyoglobin fluoride andl metmyoglobin azide. -The extreme g factors for each derivative are listed in Table I. TABLE I , Derivative Group attached gJ` ~r,in Bohr magnetons Acid methaemoglobin H20 - 2.0 6. 0 ' 5.84 Acid metmyoglobin HZO 2.0 6.0 5.85 Metmyoglobin fluoride .F 2.0 6. 0 5.92 Metmyoglobin azic'e N3 2.8 1.70 2.84 The parallel suffix refers to directions parallel to the axis through the iron atom normal to the haem plane and the perpunclicular suffix to directions at right angles to this axis (i. e. , in the hacni planeX It can be seen from Table I- that the four derivatives so far studied are di vided into two classes by both 1003546917

the resonance and susceptibility measurements. As a result of the suscep#i- bility data these two groups were clasbified' as "essentially ionic'"' and '!essentially covalent" corresponding to the cases of a spin quantum number = 5/2 or 1/2. Thus, if ionic binding is assumed, the central positively charged ferric ion will be left with five 3d electrons. These will line up with their spins parallel according to Hund's rule and so half fill each of the five vacant 3d orbitals. A total electronic spin moment of 5/2 is thus formed which will itself be quantized' in any applied field. On the other hand, if covalent bonding takes place six pairs of electrons are required to form with the surrounding ligands. These electrons donated by the surrounding atoms will occupy certain orbitals of the ferric iron, anu it can be shown that the most stable configuration for octahedral bonding is the one in which two 3d orbitals and the one 4s orbital and three 4p orbitals are employed. The five non bonding 3d electrons of the ferric iron are therfore, left with only three 3d orbitals to occupy and hence four are forced to pair leaving one unpaired and a resultant S = 1/2. If this simple interpretation were correct the resonance spectrum obtainc:d from the essentially ionic conzpounds would be expected' to have the same genoral features as that of the other ionic ferric salts. Thus, the total spin and associated magnetic moment corresponding to the configuration with S- 5/2 would take up six different orientations varying from +5/2 to -5/2. Transitions can then be induced between each of these levels according to the selection rulep,S;±li and thus five electronic absorption lines would be expected centered on a g factor of 2. 0. However, the work on single cryEtals of 1003546918

'' acid mEthaemoglobin, metmyoglobin and metmyoglobin fluoride showed that the g factor was in fact, anisotropic varying from a g// of 2. 0 to a g-L of 6. 0, and that there was only one electronic transition observable. . A theoretical explanation of the observed anisotropic values has been provided by assuming that very strong asymmetrical crystalline fields This highly anisotrdpic g factor of the "essentially ionic" haemo- globin derivatives makes the detection of the pararr,agnetic resonance signal are active on the iron atoms so that the energy difference between the six different orientations of the total spin vector of 5/'2 are much larger than of amorphous samples difficult, since the resonance line extends from approximately 1300 gauss.to 3250 gauss. This of course, accounts for the each of any apparent signal from the sample of untreated rabbit lungs. In contrast to the large variation from g = 2 to g = 6 observed for the ionic derivatives the azide derivative which is "essentially covalent" has g, factor spread across the free spin value, indicating an S of 1/2 with considerable orbital interactions. The signal we observe from rabbit lungs which have been treated with tobacco smoke is als o located at approximately the free spin value. Consequently, we have tentatively attributed at least part of our observed signal to "essentially covalent" derivatives of haen.o- globin being formed by reaction of the normal hatrro;lobin with constituents 0 of the tobacco smoke. 1003546919 So far the above discu9aion o the nature of the resonance is hypothesis based upon what is known about iron in other complexes and ha:

_ , . ...:. :. . ,..,,,,;. to be verified.experimentally. F'owever, assuming the hypothesis is correct, such a complex provides a vehicle for carrying the chemicals present in cigarette smoke to the liver'for the production of for example, acetonitrile.

resonance signal is in fact as we have outlined. 1. To establish that the origin of our observed electron spin • PROPOSED PROGRA1v1 . To determine the types of molecules and free radicals that will react with haemoglobin to produce the type of complex we have ob- . served from the reaction of tobacco smoke with haemoglobin. In this in tobacco smoke, smog, and automobile exhaust.- Since all three systems study we will restrict ourselves to compounds which are normally found contain many conUtituents in common, at this stage we will restrict our- selves to.the investigation of representative examples of these common species. is the complex once formed stable or is the decomposition of the complex to reform undamaged haemoglobin:a probable reaction? 4. To determine what effect the formation o. such a comp,ex has 3. Having established the necessary requirements for the pro- duction of such a complex, to investigate the stability of the complex, i. e. , on the general reactivity of the haemoglobin molecule. Since the bonding of the iron atom to both the porphyrin ring and~ the globin molecule changes upon formation of such a complex this is accompanied by a change in electron distribution throughout the whole molecule. Such a change in electron distribution will necessarily cause changes different areas of this complex molecule. ~ 0 in the chemical reactivities of CA FL~ ~ ~ ~"~

SUPNISSIO I OF REPORTS A bi-annual report will be submitted to the Tobacco Research Council throughout the course of this program, with a final report summarizing the entire program. It is anticipated that much of this work will be appropriate for publication in the open Scientific Literature, but prior to publication the Sponsor requested to review the manuscript and give his consent to the publication.

REFERENCES Ingram, Free Radicals as Studied by Electron Spin Resonance, 0 Academic Press, (1958). Swartz, et al, Biochem. and Biophysical Res. Commun., 21:61 (1965).