Free radicals, lipids and protein degradation

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TIBS 11 - January 1986

Free radicals, lipids and protein degradation Simon P. Wolff, Anthony Gamer and Roger T. Dean Primary oxygen radicals produced in cells and their secondary lipid radical intermediates can modify and fragment proteins. The products are often more susceptible to enzymatic hydrolysis and so radical fluxes may accelerate proteolysis inside and outside cells.

Metabolism involves the ordered transport of electrons from reducing substances to oxidizing species and its linkage to energy conversion. In aerobic organisms, the terminal electron acceptor is oxygen, which receives four electrons in a concerted fashion to produce water. Single electrons may, however, 'leak' at sites of transfer (e.g. active sites of oxidoreductases and the mitochondriai electron transport chain), permitting the inappropriate, single electron reduction of oxygen to the radical (characterized by the presence of an unpaired electron) superoxide (.02-). This radical may disproportionate or accept a further electron from a reducing agent such as thiols or ascorbate to yield peroxide (H202). There is in vitro evidence that H202 may then react with certain chelates of copper and iron (e.g. Cu/histidinyl complexes) to yield the hydroxyl radical (-OH) 1. The hydroxyl radical can react with most molecules very rapidly by a combination of addition, hydrogen abstraction or electron transfer reactions; this makes it an attractive candidate for the cytotoxic action of radicalgenerating species in vivo. Unfortunately, the very high reactivity of -OH imposes theoretical and practical restraints on its detection from such systems in vivo 2. Production of .OH in vitro is usually demonstrated by competition studies. A very high concentration of some 'scavenger' which reacts with .OH rapidly is incorporated into the system and its ability to protect some 'target molecule' indicates that .OH is indeed produced and responsible for the damage observed. An extension of that approach is the electron spin resonance (ESR) technique of 'spin trapping '2 in which the radical scavenger (spin trap) reacts with .OH to yield a stable and characteristic radical (spin adduct). Confirmation that -OH has actually been S. P. Wolff and R. 1". Dean are in the Cell Biology Research Group and A. Garner is at the Department of Biochemistry, Brunel University, Uxbridge, Middlesex UB8 3PH, UK. Correspondence to R.T.D.

trapped is achieved by examining the ability of further .OH scavengers to decrease the concentration of the hydroxyl radical spin adduct detected. Biological systems, however, contain high concentrations of molecules that act as intrinsic radical scavengers; thus, whereas it may be possible to employ further radical scavengers and produce some inhibition of a 'hydroxyl radical' effect (for example, ethanol and dimethylsulphoxide can inhibit alloxan-induced diabetes in mice3), it is difficult to use spin traps at concentrations sufficient to trap enough •OH for detection by ESR. One of our hypotheses is that secondary radicals, though less reactive, may be as important as .OH in the biological actions of free radicals. Despite the formidable problems of demonstrating the formation of radicals in vivo, their existence is suggested strongly by the existence of the enzymes superoxide dismutase and catalase (which decompose . 0 2- and H 2 0 2 , r e s p e c t i v e l y ) . The presence of these enzymes suggests that the catalysed removal of these partially reduced oxygen species is biologically advantageous and that their production is an unavoidable consequence of normal metabolism. Indeed, certain white blood cells generate •0 2- deliberately, by means of a specialized membrane-bound NADPH oxidase and this may participate in the killing of microorganisms and tumour cells4. Several other factors may increase the rate of radical production in vivo. These include (1) increases in the amount of 'decompartmentalized' iron and copper 1 leading to the 'auto-oxidation' of reducing agents such as thiols, ascorbate, reduced nucleotides and monosaccharidesS; (2) accumulation of species (such as quinones) which can establish redox couples with cellular reductants; and (3) the activation (by reduction with one or two electrons) of xenobiotics by enzymatic systems. Radical production thus probably occurs at most intracellular sites (e.g. in the endoplasmic reticulum and lysosomes; Dean et al. in

Ref. 6) as well as at surface sites of some types of cell. The suspicion that radicals are generated in vivo now needs to be corroborated by coupling their known effects on macromolecules in vitro to changes in molecules extracted from cells that have been exposed to some factor thought to generate radicals. There is a great deal of radiobiological literature on radical interactions with nucleic acids, and these interactions have recently been coupled to the demonstration of expected oxidation products in human urine. Hydroxyl radicals are known to fragment polysaccharides (e.g. hyaluronic acid) and free radicals participate in lipid peroxidation7. In contrast, little is known of the interactions of free radicals with proteins. Proteins are present inside and outside cells in very high concentrations and, because many are catalytic, modifications by free radicals may have an amplified effect. Proteins may thus be critical targets. Modification of protein activity by free radicals The modification of amino acid residues b y . O H and.O2-, and subsequent changes in enzyme activity, have been used to identify residues crucial for protein function, such as methionine, tryptophan, histidine and sulphydryl groups2, s. More recently, enhancement of inactivation of certain enzymes by secondary radicals, such as thymine peroxy radicals, has been describedL In general, the consequence of radical modification of enzymes is inactivation. However, activation of some enzymes may occur, for example, by inactivation of an enzyme inhibitor. Thymine peroxy or -OH radicals can inactivate ct-l-proteinase inhibitor concomitant with methionine oxidation2,9 Repair mechanisms may exist for several of the protein modificationsl°. Cellular reductants and antioxidants may replenish electron or hydrogen atom 'holes' after -OH attack and cleave protein-disulphides. Some enzymes (e.g. methionine sulphoxide reductase 11) can repair stable oxidation products. Protein crosslinking and fragmentation Free radicals such as -OH and possibly alkoxy (RO.) intermediates of lipid peroxidation (see below) can fragment and crosslink protein 12-13. In the absence of molecular oxygen,.OH induces crosslinks in protein which are often resistant to reduction (and are thus not solely disulphide bonds), such as dityrosine1. Some crosslinking may also

1986,ElsevierSciencePublishersB.V., Amsterdam 0376- ~67/86,'$02.00

28 occur in the presence of oxygen but fragmentation is then much more pronounced. Steady-state gamma radiolysis permits 2.~° generation of defined free radicals and shows that .OH in the presence of molecular oxygen (-OH/O2), fragments many proteins efficiently (Dean et al. in Ref. 6) whereas peroxy radicals (ROO.), including . 0 2- and its conjugate acid, the hydroperoxy radical (HO2.), are inert in this respect.

T I B S 11

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January 1986

age may be a significant determinant of synovial degradation (Fig. 3). R

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Interactions between httermediates in lipid peroxidation and proteins Food technology studies have shown that peroxidizing lipid damages proteins. Most emphasis has been placed on pro"OH/02 tein crosslinking but there is also evidence of fragmentation22. These reactions with proteins may involve both the Proteins are fragmented and modified radicals and the aldehydes generated R by H202 in the presence of transition during lipid peroxidation7 (the latter I metals or suitable chelates thereof i3,15,16. perhaps being involved in crosslinking). - - C - - N - - C - - C - - N - - ~ x - C a r b o n peroxy I] I / II I radicals Lipid hydroperoxides (ROOH) are Chelated iron must possess an H202O H 0"20 H stable intermediates of peroxidation (and coordination site for this reaction to occur. Hydroxyl radicals so genaccumulate in peroxidized lipid) which erated may react with the chelating can react with transition metals generatspecies rather than escape from the site ing RO- and ROO. radicals. These may of their generation. Thus generation of react with protein closely associated with •OH by copper/histidinyl complexes the peroxidizing lipid. It is interesting seems to lead to oxidation of the that hydroperoxides are also formed on R histidine residue to yield aspartate 17. proteins during radical attack but they I are a very minor product. ~-C--N=C--C--N-Iminopeptide Protein fragmentation by -OH is a II II / intermediates selective process: fragments of defined Perhaps the most widely studied O O I:1 rather than random length are generbiological lipid/protein system is that of ated13A5,16. The number of fragments low-density lipoprotein (LDL). During Mild acid hydrolysis produced varies from protein to protein, peroxidation of the lipid component, collagen giving a large number of fragthe apoprotein of LDL becomes fragmented 23 and there is crosslinking ments. Attack and modification by -OH Products mC NH4~ R m C - - C - - N - is expected to depend on the relative and residue modification. Two stable products of lipid peroxidation, malonconcentrations and bimolecular rate constants of the reaction of the amino acid aldehyde and 4-hydroxynonenal can side chains with this radical. However, modify lysine residues of the Apo-B by although the initial site of .OH attack Fig. 1. Reaction scheme for model polypeptides an addition reaction. In the case of modimay be random, there can be very rapid proposed by Garrison. Hydroxyl radical-mediated fication of 4-hydroxy nonenal, LDL intra- or intermolecular hydrogen/elec- scission of model polypeptides occurs via hydrogen loses its affinity for the classical fibroblast tron transfer which tends to locate the abstraction at the eL-carbonfollowed by peroxy rad- receptor (Jessup et al., unpublished). A free electron (spin centre) at the amino ical formation. This radical decomposes (by hyd- prolonged half-life in the circulation may acid residue to its lowest free energy roperoxy radical elimination) to form the iminopep- result, permitting increased uptake tide. Under conditions of mild hydrolysis these destate18,19. Molecular oxygen may inhibit compose to yield acid, ammonia and dicarbonyl by macrophages and subsequent atherosclerotic foam-cell formation (see this 'repair' process and thus the residues compounds. contribution by Yagi, p. 18 of this issue). modified may not be those found under oxygen-depleted conditions. FragmentaThere is much evidence7 of inactivation increases the number of amino attack on polyproline17. Our proposed tion of membrane enzymes during lipid groups 16, suggesting that cleavage in- mechanism accounts for the generation peroxidation but the mechanisms volves peptide-bond hydrolysis. This of the new amino termini and, most involved are not known. Because the contrasts with the oxidative scission reac- importantly, predicts the generation of mitochondrion is an important source of tion proposed earlier8 in which deamina- new N-terminal glutamate residues. The cellular -02- and H202, we have studied tion is involved (Fig 1). reactive oxygen species -OH/O 2 is not interactions between mitochondrial proThere is evidence that fragmen- the only one able to fragment protein; teins and free radicals derived either tation occurs by oxidation and sub- hypochlorous acid (HOCi), from the from electron transport or from lipid sequent spontaneous hydrolysis of proline action of myeloperoxidase on H20 2 and peroxidation. We used mitochondrial residues, for which we suggest a chloride ion, can also cleave peptide membrane monoamine oxidase, radioactively-labelled with the irreversible mechanism (Fig. 2). The lengths of the bonds20. We have observed that (radiolytic) inhibitor pargyline, as an intrinsic fragments produced from bovine serum albumin (BSA) are as would be expected •OH damage within whole cartilage target of radical damage (Dean and from cleavage at proline residues 12, and occurs at the polypeptide core of the Garner, unpublished). Radicals generthe disappearance of proline is assoc- proteoglycan rather than on the ated homogeneously by radiolysis were iated with a stoichiometric increase in glycosaminoglycan21. This may be due to much less efficient in fragmenting proglutamate concentration (Ref. 18 and the greater reactivity (-100-fold) of tein in mitochondrial membranes than in our unpublished data). There is also an •OH with proteins than with polysac- free solution. However, fragmentation increase in glutamate concentration and charides and suggests that radical dam- was produced not only by -OH/O 2 but a decrease in viscosity during radical age to the protein constituents of cartil- also by HO2-. This fragmentation was inO H

H

29

T I B S 11 - January 1986

I•C:O

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protein modifications. Hydroperoxides in damaged proteins do not undergo such a reaction to any significant degree during a two-phase experiment.

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H202; since transition metals are present as impurities in most laboratory reagents, these effects probably involve the action of transition metals on H20 2 to give species which could damage the proteins in a similar manner24. Similarly, many data imply that -SH oxidation (due to radicals or otherwise) increases hydrolytic susceptibility6. These observations suggest that if generation of reactive radicals occurs significantly in vivo, one consequence may be the accelerated enzymatic hydrolysis of damaged proteins.

Even limited oxidative modification of certain proteins can render them more susceptible to enzymatic hydrolysis. Thus, systems which generate oxygen radicals inactivate glutamine synthetase by oxidizing an active-site histidine (perhaps in a copper-mediated reaction) and the oxidized protein is much more susceptible to many different proteinases (Levine in Refs 6, 11; Rivett in Ref. 6). Conformational changes may also be involved in this process and we find 14 that about two radical events per molecule of BSA suffice to cause conformational changes detectable in fluorescence spectra and increases in susceptibility to proteinases. This increased hydrolytic susceptibility can be induced by .OH, in both the presence and absence of 02, as well as by a variety of peroxy radicals (which cause no change in protein molecular weight) but not by .02- , at least in BSA. Thus, both fragmenting and crosslinking conditions can induce increased susceptibility. Several connective-tissue components and haemoglobin, become more susceptible to enzymatic hydrolysis after exposure to

Does radiral damage influence protein breakdown in vivo? Few studies have approached this problem, either directly or indirectly, but some data exist on endogenously generated radicals in chloroplasts and mitochondria. Mitochondriai degradation during reticulocyte maturation, seems to depend on radicals produced by an endogenous lipoxygenase25 Furthermore, we found that isolated rat liver mitochondria in. State 4 (limited by availability of ADP) degrade their endogenously synthesized protein faster when radical fluxes are enhanced (by chain blockers and uncouplers) than when they are minimized26. It will be interesting to observe the effects of the mitochondrial uncoupling protein in

peptide

Fig. 2. Proposed reaction scheme for cleavage at proline residues. In proteins, hydroxy radicalmediated scission seems to occur preferentially at proline residues. In this case, hydrogen abstraction occurs at the gamma-carbon leading, via a peroxy radical, to a 2-pyrrolidone intermediate. This undergoes spontaneous hydrolysis, under physiological conditions, at the peptide bond. Subsequent mild hydrolysis reveals a new N-terminal glutamate residue.

hibited by .OH scavengers as well as by inhibitors of lipid peroxidation, such as the vitamin E analogue Trolox C. There was also much lipid peroxidation during radical attack. When the products of such radiolytic radical attack were exposed to transition metals, there was more fragmentation of protein, particularly at low pH. At neutral pH, a complex triphasic reaction ensued, which involved both fragmentation and crosslinking. These results suggest that the radical attack led to direct fragmentation of intramembrane protein as well as the formation of lipid R O O H which could then decompose in the presence of transition metals to produce further

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Fig. 3. Synergistic cartilage degradation by free radicals and hydrolases. Phagocytes release hydrolases, reactive oxygen species (R. O.S.) such as superoxide and transition metal polypeptide chelates. The latter components interact generating damaging free radicals. These may fragment protein directly or cause it to become more susceptible to hydrolase attack and endocytic uptake. The collagen triple helix, and the proteoglycen monomers (brush-structure) associated with the hydrochloric acid chain (open circles), are shown as typical substrates.

30 brown-fat cells on radical fluxes and proteolysis. Chloroplast 32 kDa protein is also degraded more rapidly when radical fluxes are enhanced by illumination27. In a few cases, exogenous agents which may increase radical generation are known to enhance proteolysis; for instance, phenylhydrazine increases breakdown of haemoglobin in reticulocytes28. The interpretation of such studies is difficult, however, because all the manipulations involved have effects on redox state and ATP concentration. Cellular proteolysis requires ATP and so changes in ATP concentration can influence proteolysis6. It is interesting that depletion of cellular reductants (such as N A D H and glutathione (GSH)) is often associated with enhanced proteolysis6. One explanation might be decreased repair of radical damage to proteins under conditions of oxidative stress. Similarly, zinc is known to possess some antioxidant properties and there can be an inverse correlation between zinc status and proteolytic rate. Again, it may be that zinc status affects proteolysis through some other pathway. More specialized antioxidants such as I]-carotene and vitamin E may have a more critical role in retarding radical-induced proteolysis, especially that involving lipid intermediates. In spite of the general indications that radical damaged proteins are degraded rapidly in vivo, some polypeptides which can undergo radical modification accumulate within cells. For example, lipofuscin (an aggregate of peroxidized lipid and proteins) accumulates in lysosomes of aged cells, Alzheimer's disease brain cells, and iron-overloaded hepatocytes. The derivation (proteolytic? radical-degraded?) of the polypeptides is not known, nor is their rate of degradation. Rates of turnover of oxidized protein might also be a factor in the pathogenesis of cataract. This disease involves the accumulation of aggregates of conformationally-altered protein, containing oxidized cysteine and perhaps oxidized methionine residues, which may be responsible for light-scattering and lens opacification. Opaque regions also contain peptides of unknown originz9. We find that free radicals fragment bovine lens protein, produce disulphide-bonded aggregates and render the protein more susceptible to enzymatic hydrolysis. Thus free radicals can mimic the changes seen during cataractogenesis. Lens protein turnover in vivo (3% per month) is unusually slow and we suggest that it may be restrained by the gel nature of the lens. In such gels, water activities are

TIBS 11 - January 1986

lower than in normal cytosol and so hydrolytic enzymes (such as proteinases) are both less active and less able to reach their substrates. Specialized degradative lyases (cleaving without use of water) might be involved but in any case the limitation on hydrolysis would apply equally to radical-damaged proteins, and hence they might remain in detectable quantities. The oxidized light-scattering aggregates in cataracts possess fewer free lysine groups and many of the agents able to cause cataract are both radicalgenerating and lysine-modifying. Whilst oxidation to proteins may be an unavoidable consequence of normal metabolism, which is countered by efficient and protective proteolysis, unusually high rates of oxidative damage to proteins and/or inhibition of subsequent proteolysis may lead to the accumulation of denatured protein which finally impairs tissue function, particularly in a situation of pre-existent restricted proteolysis, such as the lens. This may be of relevance to foam-cell formation, lipofuscin accumulation and Heinz body formation in erythrocytes, as well as to cataractogenesis. In extracellular proteolysis, for example in cartilage degradation in rheumatoid arthritis (Fig. 3) an attractive hypothesis is a destructive synergy between radical-mediated polypeptide damage and hydrolase attack. Here, fragments of proteins such as collagen need to be removed from the structural array of the cartilage before they can be extensively degraded. (A similar problem applies to the myofibrils in muscle.) Radical attack might produce such fragments, which may also be more rapidly endocytosed than the native molecules, thus increasing intracellular digestion. This is anticipated because adsorptive endocytosis (in which the substrate enters cells bound to the surface, rather than in solution) seems to be dictated by the same structural features of proteins which dictate susceptibility to proteolysis. Several workers have used model systems of phagocytes, such as macrophages and neutrophils, and monolayers of connective-tissue macromolecules to approach these questions. From such studies, it is clear that some of the cell types involved can create an extracellular microenvironment of low pH and restricted protein movement, in which degradation might be enhanced. In this site, radicals generated by the surface oxidase system of the cells may contribute to degradation. There is also secretion of transition metal-polypeptide

chelates (such as lactoferrin), which may catalyse the generation of -OH from hydrogen peroxide, as well as mveloperoxidase, which generates HOCI from the hydrogen peroxide Similarly, protective enzymes (e.g. superoxide dismutase) and antioxidants may be consumed. Oxidation may also inactivate proteinases and their inhibitors within the tissue: this action might either amplify or reduce the damage according to the particular circumstances30. Whether radical damage to proteins in vivo has a positive influence on the rate of proteolysis in cells and their immediate exterior remains undecided. There are of course many other critical aspects of the damage to proteins effected by radicals which may apply in vivo.

Other possible consequences of damage to proteins by radicals Most data on radical damage to biological macromolecules concern the effects of radiation, mainly on nucleic acids (because of the possible genetic effects) and lipids. Yet, for the reasons given above, particularly the catalytic role of many proteins, we suggest that damage to proteins may also be important, at least in the short term and probably in the longer term because proteins regulate gene expression. The extreme case of the possible relevance of protein damage is in cell lysis. Free radical fluxes can lyse cells. 'Professional phagocytes' generate such a flux at the cell surface in killing bacteria and parasites. We thus suggest that the critical targets of such a mechanism, might be transport proteins in membranes. A limited radical attack on a critical transport protein might cause a greatly amplified effect on the ionic homeostasis of the cells, with resultant osmotic lysis. Radical attack on some cell types results in damage to surface proteins2,3. In pathological situations in which macrophage activation occurs, phagocytes are attracted to sites of immunological activity and there is an accelerated rate of killing invading organisms. This enhanced killing involves increased secretion of both radicals and hydrolases but this may also cause accelerated degradation of extracellular macromolecules of the host, as in demyelinating diseases, arthritis and other chronic inflammatory conditions. There are also nutritional circumstances in which radical damage to proteins may be accentuated (e.g. Zn deft-

31

T I B S 11 - January 1986

ciency and kwashiorkior, which affect many people in Asia and Africa). Amongst possible reasons for this are: (1) decompartmentalization of transition metals, (2) lack of Zn or other metals which compete with iron and copper in oxidation-related transition metal reactions and (3) a lack of cellular reductants and antioxidants. In these circumstances, radical/hydrolase synergy may be of critical importance in the development of several of the pathological features. Summary We propose that radical damage to proteins may lead to enhanced enzymatic proteolysis in tissues. This process may be an adapted feature of normal metabolism but could be implicated in pathophysiology under certain circumstances. First, excessive rates of radical damage to intracellular proteins and/or inhibition of the proteolysis of oxidized protein may lead to the intracellular accumulation of denatured protein 'junk', which may interfere with cell function. Secondly, radical generation at inappropriate extraceUular sites may lead to pathological tissue degradation. Finally, we suggest that a primary target in radical-induced cell death may be membrane transport proteins. Acknowledgements We acknowledge with thanks support from the A F R C and Arthritis Council for Research, UK. References 1 Halliwell, B. and Gutteridge, J. M. C. (1984) Biochem. J. 219. 1-14; see also Anon, (1985) Lancet 143-145 2 Methods Enzymol. 0984) Vol. 105 (many articles) 3 Clark, I. A. Medicinal Research Reviews (in press) 4 Segal, A. W. 0984) Med. Biol. 62, 81-4 5 Wolff, S. P., Crabbe, M. J. C. and Thomalley, P. J. (1984) Experientia 40,244-246 6 Khairallah, E. A., Bond, J. S. and Bird, J. W. C. (eds.) 11984) Intracellular Protein Catabolism, pp. 1-702, Alan R. Liss 7 Slater, T. F. (1984) Biochem. J. 222, 1-12 8 Garrison, W. M. (1968) Curr. Top. Radiat. Res. 4, 43-94 9 Gee, C. A., Kittridge, K. J. and Wil[son, R. L. (1985) Br. J. Radiol. 58,251-256 10 WiUson, R. L. (1983) in Radioprotectors and Anticarcinogens (Nygaard, O. F. and Simic, M. G), pp. 1-22, Academic Press 1l Methods EnzymoL (1985) Vol. ! 11 12 Schuessler, H. and Schilling, K. (1984) Int. J. Radiat. Biol. 45,267-287 13 Wolff, S. P. and Dean, R. T. Biochem. J. (in press) 14 Amado, R., Aeschbach, R. and Neukon, H. (1984) Methods Enzymol. 107. 377-388 15 Orr, C. M. W. (1967) Biochemistry 6, 3001)-3006

16 Gutteridge, J. M. C. and Wilkins, S. J. (1983) Biochim. Biophys. Acta. 759, 38-41 17 Cooper, B., Creeth, J. M. and Donald, A. S. R. (1985) Biochem. J. 228, 6154526 18 Levitzki, A. and Anbar, M. (1967) J. Am. Chem. Soc. 89, 4185-9 19 Butler, J., Land, E. J., Swallow, A. J. and Prutz, W. (1984) Radiation Phys. Chem. 23, 265-270 20 Thomas, E. L. 11979) Infect. Immun. 23, 522-531 21 Dean, R. R., Roberts, C. R. and Forni, L. G. (1984) Biosci. Repts. 4, 1017-1026 22 Schaich, K. M. (1980) Crit. Rev. Food Sci. Nutr. 13, 89-130; 131-160; 189-244 23 Parthasarathy, S., Steinbrecher, U. P., Barnett, J., Witzum, J. L. and Steinberg, D. (1985) Proc. Natl Acad. Sci. USA 80, 3000-3004

24 Fligiel, S. E., Lee, E. C., McCoy, J. P., Johnson, K. J. and Varani, J. (1984) Am. J. Pathol. 115,418-425 25 Rapoport, S., Schmidt, J. and Prehn, S. (1985) FEBS Lett. 183, 370-374 26 Dean, R. T. and Pollak, J. K. (1985) Biochem. Biophys. Res. Commun. 126, 1082-1089 27 Matto, A. K., Hoffman-Falk, H., Marder, J. B. and Edelman, M. (1984) Proc. NatlAcad. Sci. USA 81,4070--4075 28 Goldberg, A. L. and Boches, F. S. (1982) Science 215, 1107-1109 29 Ciba Foundation Symposium 106 'Human Cataract Formation', (1984 L pp 1-284, Pitman 30 Weiss, S. J. and Regiani, S. (1984) J. Clin. Invest. 73, 1297-1303

Biochemical explanations for folk tales: vampires and werewolves According to David Dolphin, a biochemist at the University of British Columbia, vampires and werewolves may really have existed in the Middle Ages. The vampire condition, however, would have been passed on in genes rather than by a bite on the neck. Congenital erythropoietic porphyria is an autosomal recessive disease caused by a deficiency of the enzyme uroporphyrinogen III cosynthetase in the pathway of haem biosynthesis. Not enough urophorphyrinogen III is formed, and instead large quantities of its isometric isomer, uroporphyrinogen I, are synthesized. This isomer has no physiological activity, and it is secreted in large quantities: the urine becomes red. There is also reddishbrown pigmentation of the teeth, which fluoresce strongly orange-red under UV. Not surprisingly, erythrocytes without their correct haem composition are destoyed prematurely, and the unfortunate person suffers from hemolytic anaemia. But worse is to come; the skin becomes unusually sensitive to light, singlet oxygen giving rise to mutilating skin lesions, which may be so bad as to cause the loss of nose and fingers. The condition also causes hypertrichoses, an excessive growth of hair. So imagine an excessively, hairy individual, very pale, with skin deformities, and an aversion to light. The lips and gums may be taut, giving the appearance of fangs (red fangs!). It

has been suggested that such symptoms may have given rise to the old folk lore surrounding werewolves. Nowadays, the condition can be treated by injections of haem; but in the Middle Ages, the victim's only chance may have been to drink lots of blood, which may have given rise to the vampire tales. Why Transylvania? In the Middle Ages, in such a remote mountain area, there would have been a lot of inbreeding and a high risk of congenital defects. (Presumably this scenario was not confined to Transylvania!) What of the supposed protections against vampires? While there is no doubt that a stake through the heart of someone suffering from porphyria would kill, there is also some basis for the protective effects of garlic - although the vampire would have to eat it. Apparently, a chemical in garlic (dialkyl disulphide) may interfere with a group of liver enzymes, the cytochromes /'450, which are haem proteins. It has recently been suggested that dialkyl disulphide may modify the cytochrome P450 haem, and that the haem complex soformed can act as an inhibitor of the last step in haem biosynthesis. The effect is harmless in most humans, but it may increase the severity of the porphyrias. So, allowing for the exaggeration of rumour, could this disease be the scientific kernel of truth in the legends of Bram Stoker? JUDITH HALL