manganese porphyrins are well characterized and stable ( collman et al , science 261 : 1404 ( 1993 ), tsang et al , inorg . chem . 29 : 2848 ( 1990 ), harriman et al , j . chem . soc . faraday trans . 275 : 1532 ( 1979 )). moreover , such compounds have been reported to catalyze the dismutation of o 2 - with rate constants as high as 4 × 10 7 m - 1 s - 1 ( farragi , m . in oxygen radicals in chemistry and biology ( bors , w ., saran , m ., and tait , d ., eds .) pp . 419 - 430 , walter de gruyter , publ ., berlin ( 1984 ), pasternak et al , j . inorg . biochem . 15 : 261 ( 1981 )). the present invention results from the discovery that manganic porphyrins exhibit reversible redox behavoir . more specifically , the invention results from the finding that mn ( iii ) 5 , 10 , 15 , 20 - tetrakis ( 1 - methyl - 4 - pyridyl )- 21h , 23h - porphine tetra - p - tosylate ( mntmpyp ) in vivo is reduced enzymatically at the expense of nadph and non - enzymatically by gsh , with a rate constant for reoxidation of the reduced form by o 2 - of 4 × 10 9 m - 1 s - 1 . the compound thus acts as a nadph / gsh : o 2 - oxidoreductase in vivo . such action requires replenishment of nadph and gsh ; that can be achieved at the expense of carbon source ( eg glucose ). mntmpyp is not the only manganic porphyrin that imparts an nadph : o 2 - oxioreductase activity and acts like a gsh : o 2 - oxidoreductase . several manganic porphyrins , with substituents on the methine carbons , were prepared and examined for stability , redox behavior , catalysis of the dismutation of superoxide radical ( o 2 - ), and for the ability to protect a sod - null strain of e . coli against dissolved oxygen , and a sod - competent strain against paraquat ( see example that follows ). all of the compounds tested exhibited reversible redox behavoir and were stable to edta in both the oxidized and reduced states , and several were able to catalyze the dismutation of o 2 - with rate constants of . sup .˜ 10 7 m - 1 s - 1 . the marked protective effects of certain of these compounds exceeded that which could be anticipated on the basis of such rate constants . while the present invention is described with reference to manganic porphyrins bearing substituents on the methine bridge carbons , other metals can also be used . examples of such metals include iron ( fe ( iii )→ fe ( ii )), cobalt ( co ( iii )→ co ( ii )), nickle ( ni ( ii )→ ni ( i )) and copper ( cu ( ii )→ cu ( i )). the complexes of the present invention can be obtained commercially or can be prepared using methods known in the art ( see also example that follows ). the oxidoreductase function of the molecules ( complexes ) of the invention permits their administration as pharmaceuticals in concentrations sufficiently low to avoid problems associated with toxicity , which concentrations are sufficiently high to achieve the effect sought . appropriate doses can be readily determined by one skilled in the art , given the present disclosure ( as an example , a dose of 1 - 50 mg / kg can be used ). as regards the formulation of the molecules of the invention as compositions and as regards methods of using the molecules of the invention ( eg in autoxidation inhibition and in protection ), see application ser . no . 08 / 089 , 813 ( the entire contents of which is incorporated herein by reference ) ( see also u . s . pat . no . 5 , 223 , 538 and u . s . pat . no . 5 , 227 , 405 ). the invention is illustrated by way of reference to the non - limiting example that follows . preparation of mn porphyrins -- complexes of h 2 - tmpyp and h 2 - tmap , with mn , fe or co , were prepared by the method of pasternack et al , biochemistry 22 : 2406 ( 1983 ). metal ligation was followed spectrophotometrically . the mntmpyp complex was characterized in terms of its soret band at 463 nm , e m = 9 . 2 × 10 4 m - 1 cm - 1 ( harriman et al , j . chem . soc . faraday trans . 275 : 1532 ( 1979 ); pasternack et al , biochemistry 22 : 2406 ( 1983 )). the soret band for the mntmap complex was at 465 nm with e m = 9 . 2 × 10 4 m - 1 cm - 1 . as judged by constancy of their absorption spectra , these compounds were stable from ph 1 - 9 . moreover , the mn - porphyrins did not oligomerize in the range 0 - 3 μm , since there were no deviations from beers law . the mn complexes of h 2 - tpyp and of h 2 - tbap were prepared by the method of harriman and porter ( j . chem . soc . faraday trans . 275 : 1532 ( 1979 )). for mntpyp the soret band was at 463 nm and e was 9 × 10 4 m - 1 cm - 1 ; while for mntbap the corresponding values were 468 nm and 9 . 3 × 10 4 m - 1 cm - 1 ( harriman and porter , j . chem . soc . faraday trans . 275 : 1532 ( 1979 )). assays -- catalysis of the dismutation of o 2 - was measured by using xanthine oxidase plus xanthine as the source of o 2 - and ferricytochrome c as the indicating scavenger of o 2 - ( mccord et al , j . biol . chem . 244 : 6049 ( 1969 )). rates of reduction of cytochrome c were followed at 550 nm with a beckman model du - 70 . assays were conducted in the absence and in the presence of 0 . 1 mm edta in 50 mm potassium phosphate , ph 7 . 8 and at 25 ° c . rate constants for reaction of the metal porphyrins with o 2 - was based on their competition with cytochrome c for reaction with o 2 - , using k o2 - , cyt c = 3 . 0 × 10 6 m - 1 s - 1 ( koppenol et al , israel j . chem . 24 : 11 ( 1984 )). possible interference by inhibition of the xanthine oxidase reaction by the test compounds was examined by following the rate of accumulation of urate at 295 nm in the absence of cytochrome c . the oxidation of nadph was followed at 340 nm . anaerobic measurements were made using an anaerobic cuvette ( hodgson et al , anal . biochem . 51 : 470 ( 1973 )) purged with n 2 which had been passed over a bed of hot copper foil to remove traces of o 2 and which was conducted to the cuvette through copper tubing . o 2 uptake was followed polarographically with a clark electrode . all measurements were at 25 ° c . culture of e . coli -- rates of growth of cultures of e . coli were followed turbidimetrically at 700 nm to minimize interferences from the absorbance of test compounds . culture media were prepared as described by maniatis et al , molecular cloning , a laboratory manual , cold spring harbor press cold spring harbor , n . y . supplemented medium contained 0 . 2 % glucose , 0 . 2 % casamino acids and m9 salts in tap water , ph adjusted to 7 . 0 . minimal medium contained 0 . 2 % glucose and 0 . 5 mm each of leucine , threonine , proline , arginine and histidine , plus m9 salts in tap water , ph adjusted to 7 . 0 . in some cases mn - poor medium was prepared by substituting mops for the phosphate present in m9 and by using chelex - 100 to remove contaminating metal cations . cu ( ii ), zn ( ii ) and fe ( ii ) were then added to . sup .˜ 1 mg / l . all media were further supplemented with 30 mg / l of thiamine and of d - pantothenic acid . when added to media , test compounds were filter - sterilized . extracts of cells grown in m - 9 medium supplemented with 0 . 2 % casamino acids were prepared , after washing with 50 mm sodium phosphate at ph 7 . 3 and resuspension in this buffer , by use of the french press . the lysate was clarified by centrifugation . lb adjusted to ph 7 . 0 medium was prepared as described ( maniatis et al , molecular cloning , a laboratory manual , cold spring harbor press cold spring harbor , n . y .). protein was measured by the method of lowry et al ( j . biol . chem . 193 : 265 ( 1951 )). electrochemical characterization of test compounds -- cyclic voltammetry was performed using a par model 175 potentiostat and a par model 173 programmer . the working electrode was a polished pt button electrode pretreated by sonication in 1 n hcl . an ag / agcl electrode ( bioanalytical systems ) served as the reference (- 200 mv vs nhe ), and a 1 mm diameter pt wire auxiliary electrode . cyclic voltammetry was performed in 0 . 05 m phosphate buffer , ph 7 . 8 , 0 . 1 m nacl , 21 ° c . e 1 / 2 values were obtained by taking the average of the e pc and e pa values . typical scan rates were from 100 to 500 mv / sec . the electrochemistry was performed in argon purged solution with added na 2 s 2 o 4 to scavenge trace o 2 . the added na 2 s 2 o 4 had no influence on the e 1 / 2 values obtained for the mn porphyrins , but increased the current response at the electrode surface by reducing the competition of mn ( ii ) porphyrin with electrochemically reduced o 2 species . 1 - methyl imidazole was also present in the solution to compete for the axial coordination sites on the mn porphyrins . the 1 - methyl imidazole had no influence on the e 1 / 2 values obtained , but decreased the δe pp values ( e pa - e pc ). when 1 - methyl imidazole was added , the ph of the solution was adjusted to ph 7 . 8 using 1 n hcl . stability of mn porphyrins -- the structures , proper names and abbreviated designations for the test compounds are given in fig1 . the optical spectra of mntpyp , mntmpyp , mntmap , mntbap and mntminp were independent of ph in the range 1 . 0 - 9 . 0 . these complexes were unaffected by 0 . 10 mm edta ; which represented at least a ten - fold excess of edta over the test compounds . at ph 7 . 8 these complexes could be reduced with dithionite and then reoxidized by o 2 without loss and this could be done in the presence of excess edta . it follows that both the mn ( ii ) and the mn ( iii ) forms of these complexes were stable towards edta . activity in vitro -- mntmpyp exhibited sod - like activity in the xanthine oxidase - cytochrome c assay , in the presence of a . sup .˜ 100 - fold molar excess of edta ( see fig2 ). mntmpyp , tested up to 6 μm , did not inhibit the xanthine oxidase reaction and the optical spectrum of this porphyrin was not changed by exposure to this reaction under the conditions of the assay for sod activity ( mccord et al , j . biol . chem . 244 : 6049 ( 1969 )) but with cytochrome c omitted . these results are in accord with the conclusions of faraggi ( oxygen radicals in chemistry and biology ( bors , w ., saran , m ., and tait , d . eds ) pp . 419 - 430 , walter de gruyter , publ ., berlin ( 1984 )). fe ( iii ) tmpyp has previously been noted to react with h 2 o 2 ( pasternack et al , j . am . chem . soc . 101 : 1026 ( 1979 )). the soret band of the mn ( iii ) tmpyp was similarly diminished by h 2 o 2 at 1 . 0 or 10 . 0 mm . however , when [ h 2 o 2 ] was only 0 . 1 mm the bleaching of the soret band of 12 . 5 μm mn ( iii ) tmpyp was very slow ; being less than 1 % loss of absorbance per minute at 25 ° c . it follows that attack by h 2 o 2 would be insignificant in catalase - proficient cells . inhibition of cytochrome c reduction by mntmpyp , when plotted according to sawada and yamazaki ( biochem . biophys . acta 327 : 257 ( 1973 )), yielded a straight line ( fig3 ) from which ic 50 was found to be 0 . 7 (± 0 . 07 ) μm and from this the rate constant for the reaction of o 2 - with mntmpyp was found to be 3 . 9 × 10 7 m - 1 s - 1 at ph 7 . 8 . this agrees very well with the rate constant of 4 . 0 × 10 7 m - 1 s - 1 determined by faraggi ( oxygen radicals in chemistry and biology ( bors , w ., saran , m ., and tait , d . eds ) pp . 419 - 430 , walter de gruyter , publ ., berlin ( 1984 )), at ph 8 . 0 , through pulse radiolysis . it appears that mn ( iii ) tmpyp acts as a sod - mimic , under the conditions of the sod assay , without causing detectable side reactions . comparable data for all of the mn - porphyrins examined is presented in table i . it should also be noted that mntmpyp was apparently unaffected by the h 2 o 2 which accumulates during the xanthine oxidase reaction since its catalytic activity was not affected by 450 units / ml of catalase . table i______________________________________results of the cyclic voltammetry and thecytochrome c assays of the mn porphyrins chargemn porphyrin ( ph 7 . 8 ) e . sub . 1 / 2 ( mv , nhe ). sup . a ic . sub . 50 ( μm ). sup . b k . sub . cat ( m . sup .- 1 . sbsp . s . sup .- 1 ) b______________________________________mntmpyp + 5 + 60 0 . 70 3 . 9 × 10 . sup . 7mntpyp + 1 c cmntmap + 5 - 100 1 . 6 × 10 . sup . 6mntminp + 5 + 44 cmntbap - 3 + 110 d______________________________________ a ) conditions : 0 . 05 m phosphate buffer , 0 . 10 m nacl ph 7 . 7 - 7 . 8 , 0 . 4 to 1 . 0 mm mn porphyrin , 0 . 5 - 1 equivalent of na . sub . 2 s . sub . 2 o . sub . 4 , 2m m 1methyl imidazole . reproducibillty of e . sub . 1 / 2 =± 10 mv . b ) conditions : 0 . 05 m phosphate buffer , 0 . 10 mm edta , ph 7 . 8 , 40 μm xanthine , ca 10 μm cyt c . sup . 3 +. reproducibility of ic . sub . 50 = ± 10 %. c ) these mn porphyrins were not active in our assay conditions . d ) mntbap interfered with the production of urate , in the presence and absence of 0 . 1 mm edta . electrochemical behavior -- cyclic voltammetry indicated reversible redox behavior for all of the mn - porphyrins examined ; as evidenced by δe pp ≦ 100 mv at scan rates in the range 100 - 500 mv / sec . moreover , in the absence of o 2 , peak currents for the cathodic and anodic waves were equivalent , also indicative of reversibility . the results of cyclic voltammetry are given in table 1 . e 1 / 2 for mntmpyp is in agreement with faraggi ( oxygen radicals in chemistry and biology ( bors , w ., saran , m ., and tait , d . eds ) pp . 419 - 430 , walter de gruyter , publ ., berlin ( 1984 )); whereas the value for mntmap obtained is 30 mv negative of the previously reported value . sodium dithionite , which was used to remove traces of o 2 , and thus to eliminate interferences from the cathodic reduction of o 2 , did not itself interfere in the potential region used during these measurements . activity in vivo -- sod - null e . coli exhibit several dioxygen - dependent auxotrophies and grow slowly aerobically , whereas they grow as well as the sod - competent strain under anaerobic conditions ( carlioz et al , embo j . 5 : 623 ( 1986 ); imlay et al , j . bacteriol . 174 : 953 ( 1992 )). as shown in fig4 either mntmpyp or mntbap increased the growth of the sod - null ji132 strain ( imlay et al , j . bacteriol . 169 : 2967 ( 1987 )) in a glucose - salts medium supplemented with 0 . 2 % casamino acids . in a simpler medium , the sod - null grew very slowly under aerobic conditions and , as shown in fig5 mntmpyp markedly increased this rate of growth . the protection effect of mntmpyp was also seen when the sod - null was grown in a manganese - deficient medium buffered with mops in place of phosphate . several mn - porphyrins were compared for their abilities to catalyze the dismutation of o 2 - ( table i ) and to increase the aerobic growth of the sod - null strain ( table ii ). their order of sod - like activities in vitro was mntmpyp & gt ; mntmap & gt ; mntpyp = mntminp = 0 . in general the sod - like activities of these complexes assayed in vitro paralleled their activity in vivo with the exception of mntpyp which was active in vivo although inactive in vitro . fetmpyp , which was active as a sod - mimic ( oxygen radicals in chemistry and biology ( bors , w ., saran , m ., and tait , d . eds ) pp . 419 - 430 , walter de gruyter , publ ., berlin ( 1984 )), was toxic at 20 μm and inhibited the growth of sod - competent e . coli . the metal - free porphyrins caused a slight growth inhibition , as did edta , probably by diminishing the availability of essential trace metals . table ii__________________________________________________________________________doublinq times for e . coli strains ab1157 and ji132in the presence of mn porphyrins none cotmpyp mntmpyp mntpyp mntmap mntbap t . sub . d ( min ). sup . a , [ concentration of mn porphyrin ] ( μm ) __________________________________________________________________________2 % supplement of ab1157 48 -- 48 [ 50 ] -- -- 48 [ 50 ] casamino acids . sup . b ji132 240 [ 27 ] 60 [ 25 ] 90 [ 19 ] 210 [ 55 ] 120 [ 37 ] 5 essential amino ab1157 130 [ 23 ] 130 [ 50 ] -- -- acids . sup . c 780 [ 23 ] 240 [ 24 ] 540 [ 10 ] -- -- __________________________________________________________________________ . sup . a ) t . sub . d was calculated as the time it takes for the optica densit at 700 nm of the sample to double during the logphase growth period . with the exception of the cotmpyp growth studies , these results are the averag of at least two trials . . sup . b ) conditions : m9 salts in phosphate buffer , tap water , 0 . 2 % glucose 2 % casamino acids , shaken at 37 ° c . . sup . c ) conditions : m9 salts in phosphate buffer , tap water , 0 . 2 % glucose 0 . 50 mm each of threonine , leucine , proline , histidine , and arginine , 37 ° c . mn ( ii ), per se , at 2 or 95 μm , did not perceptibly overcome the growth - inhibiting effect of 30 μm paraquat . in contrast , 20 μm mntmpyp eliminated , virtually completely , the growth inhibition imposed upon ab1157 ( imlay et al , j . bacteriol . 174 : 953 ( 1992 )) by 10 μm paraquat ( fig6 ). furthermore , 25 μm mntmpyp was found to eliminate the growth inhibitory effect of 20 μm paraquat and to partially alleviate the growth inhibition caused by 40 μm paraquat on the sod - competent strain . mntpyp was similarly protective , but to a lesser degree . mntmpyp was also able to overcome the effect of 5 μm paraquat on the sod - null strain . the protective effects of mntmpyp , so apparent in the amino acid - supplemented glucose plus salts medium , could not be seen in lb medium . this was due to inactivation of the mntmpyp by a component of the lb medium . reduction of mntmpyp -- centrifugation of e . coli ( ji132 or ab1157 ) which had been grown in lb containing 25 μm mntmpyp revealed that the cell pellet was red . since this is the color of the mn ( iii ) complex , it establishes that the complex is taken into the cells . in contrast , cells grown in the glucose plus salts medium , supplemented with 0 . 2 % casamino acids , were green ; while the suspending medium was red . dithionite converted the mn ( iii ) tmpyp to a green product , presumably the mn ( ii ) complex . it thus appears that mn ( iii ) tmpyp becomes reduced within e . coli and that some component of lb medium converts it to a form which cannot be thus reduced . anaerobic incubation of 25 μm tmpyp with cell extract plus 0 . 1 mm nadph , in phosphate at ph 7 . 3 or in tris at ph 7 . 8 , produced the green color and subsequent aeration restored the original red color . it follows that e . coli extract contains an nadph : mn ( iii ) tmpyp oxidoreductase . given that the mn ( ii ) porphyrin complexes can autoxidize ( oxygen radicals in chemistry and biology ( bors , w ., saran , m ., and tait , d . eds ) pp . 419 - 430 , walter de gruyter , publ ., berlin ( 1984 )), it would be expected that mn ( iii ) tmpyp should catalytically increase the oxidation of nadph by extracts of e . coli . trace 1 in fig7 demonstrates that this was the case and that sod had no effect on this oxidation of nadph . mn ( iii ) tmpyp , per se , caused a very slow oxidation of nadph and this was dramatically increased by cell extract ( trace 2 ). since 25 μm mn ( iii ) tmpyp facilitated the complete oxidation of 100 μm nadph by cell extract without any perceptible stable change in the spectrum of the complex ; it follows that the complex acted catalytically , being reduced by the nadph - dependent oxido - reductase and then reoxidized by dioxygen . thioredoxin reductase was able to substitute for cell extract in these measurements . trace 1 in fig8 shows that paraquat strongly stimulated the oxidation of nadph by thioredoxin reductase , while trace 2 demonstrates that mntmpyp was also active while the porphyrin without the manganese was not significantly active ( trace 3 ). under anaerobic conditions , the mntmpyp was converted to the green reduced form by thioredoxin reductase plus nadph . several other complexes were examined . mntpyp caused a slow oxidation of nadph and this was markedly increased by thioredoxin reductase . in contrast , co ( ii ) tmpyp and fe ( iii ) tmap rapidly and non - enzymically oxidized nadph . effect of lb medium -- the observation that lb medium elminiated both the protection by , and the greening effect of , mntmpyp : suggested that some component of this complex medium was intefering with reduction of this complex . traces 1 and 2 in fig9 show that 20 % lb in the reaction mixture inhibited the oxidation of nadph by cell extract plus mntmpyp ; whereas it had no effect when paraquat was used in place of the mn - porphyrin ( traces 3 and 4 ). it seems likely that some component of the lb medium , very likely some component of the yeast extract , converts the mn ( iii ) tmpyp to a form which is not easily reduced by nadph : mn ( iii ) tmpyp oxidoreductases , such as thioredoxin reductase . non - enzymic reduction of mn ( iii ) tmpyp -- the red mn ( iii ) porphyrin was converted to the green form by incubation with 2 - 10 mm gsh in phosphate at ph 7 . 3 or in tris at ph 7 . 8 . if the green form is indeed the autoxidizable mn ( ii ) tmpyp then mn ( iii ) tmpyp should catalyze the oxidation of gsh . trace 1 in fig1 shows that addition of 25 μm mn ( iii ) tmpyp to 10 mm gsh in phosphate at ph 7 . 3 caused an oxygen consumption the rate of which decreased during several minutes of observation . subsequent addition of additional mn ( iii ) tmpyp reinstated the more rapid o 2 consumption . trace 2 records similar effects in tris at ph 7 . 8 . it appears that mn ( iii ) tmpyp does catalyze the oxidation of gsh but that it is gradually inactivated during incubation with gsh . the effect of anaerobic incubation with gsh upon the long wavelength absorption spectrum of mntmpyp is shown in fig1 . this change in spectrum was also caused by anaerobic incubation of the compound with dithionite , or with nadph in the presence of thioredoxin reductase , or cell extract . all documents cited above are hereby incorporated in their entirety by reference . one skilled in the art will appreciate from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention . one skilled in the art will appreciate from a reading of this disclosure that mn - porphyrins are not the only compounds that function as oxidoreductases . riley & amp ; weiss have described a mn ( ii ) penta - azacyclopentadecane that catalyzes the dismutation of o 2 - with a rate constant 4 × 10 7 m - 1 s - 1 ( riley et al , j . am . chem . soc . 116 : 387 ( 1994 )). since the mn ( iii ) form of this compound is a potent oxidant , in vivo reaction is likely due to nadph and gsh , rather than to o 2 - .
the present invention relates , in general , to porphyrin complexes and , in particular , to methods of using porphyrin complexes to impart oxidoreductase activity .