IMMUNOLOGICAL ASPECTS OF URATE OXIDASE THERAPY IN HYPERURICAEMIA Department of Medicine and Therapeutics, University Col/ege, Dublin 4 D, A. Fitzpatrick~ O. FitzGerald and K. F. McGeeney Summary HE immunological relationship beTtween urate oxidase (uricase) prepared from microbiological and animal sources was studied. The enzymes from these two groups were found to be immunologically unrelated. Within each group cross reaction did occur; cross reactivity was frequent among the enzymes from animal sources. Chemical modification of pork liver enzyme did not alter the immunological integrity of the molecule even when polymerisation of the enzyme was induced. Introduction The reduction of serum uric acid by intravenous administration of a urate oxidase preparation was first attempted by London and Hudson (1957). Although no untoward reactions were noticed the drop in serum uric acid in the one gouty patient studied was not as striking as obtained with a control. This was probably due to the low level of urate oxidase activity present in the rather impure preparation used. The recent developmenl (Labcurer and Langlois, 1968) of a very active microbial urate oxidase of high purity has led to a renewal of interest in this mode of therapy (Kissel et al., 1968; Bregard et al,, 1972; Zittoun et al., 1976). A very rapid reduction of serum uric acid levels is obtained, even in the case of hyperuricaemics. Although the initial administration is unlikely to produce any detectable immune response stimulation of the immune system will almost certainly occur and subsequent injection of the same preparation may evoke antibody production The nature and extent of this anti-
body production is a function of the individual antigen used. As the immunelegy of urate oxidase has not previously been the subject of extensive study we felt it would be of interest to study the antigenic properties of this enzyme so that its clinical use can take place against a competent knowledge of the potential immunological problems which might arise. In addition the possibility of decreasing the extent of cross reaction by chemical modification of the enzyme was investigated.
Materials and Methods Antisera were produced by administering 5.0 I.U. (1 ml) of enzyme emulsified with an equal volume of Freund's complete adjuvant subcutaneously. Animals were bled after three or more injections. Pork and rat liver urate oxidase were purified by a procedure reported elsewhere (Fitzpatrick, 1972). Aspergillu~ oryzae (Botany Department, U.C.D., No. 355) was grown on minimal medium containing 0.05 per cent urate and the enzyme extracted and partially purified by ammonium sulphate fractionation. Candida utilis urate oxidase was Type IV from Sigma. L. Mahler donated the Bacillus fastidiosus enzyme and P. Labcurer the Aspergillus flavus enzyme, Ouchterlony immunodiffusion was carried out on 1 per cent agarose (Difco) plates. These were stained specifically for urate oxidase activity (Graham and Karnovsky, 1965). Enzyme inhlbi6on was studied by incubating samples of B, fastidiosus urate oxidase (0.5 ml, 0.28 I.U.) at room temperature with increas~ ing amounts of homologous antiserum for 10 minutes and assayed for enzyme
155
156
IRISH JOURNAL OF MEDICAL SCIENCE
activity. After s t a n d i n g overnight the solutions were centrifuged and the supernatants assayed for activity. All e n z y m e modifications were carried out on the presence of 0.5 per cent bovine serum albumin. Polymerisation with glu{araldehyde (0.0015 per cent) was carried out at pH 7, all other reactions at pH 10.0 in 0.1 M sodium carbonate. Treatment with h e x a m e t h y l e n e diisocyanate was as described by Synder et al. (1974), other reaction c o n d i t i o n s were those of Means and Feeney (1971). T h e modifying reagents used were, amino g r o u p s : acetic a n d s u c c i n i c anhydride, ethylacetimidate and S-ethylthioltrifluoroacetate. G u a n i d i n o groups : cycIohexanedione and phenylglyoxal. Phenolic g r o u p s : tetranitromethane. Imdazole g r o u p s : diethylpyrocarbonate. Bifunctional reagents : h e x a m e t h y l e n e diisocyanate a n d glutaraidehyde.
Results Antisera were prepared to purified preparations of ura~e oxidase from a m a m m a l i a n (pig liver) and three microbia[ species (C. ufilis, A. flavus and B. fastidiosus). Urate oxidase from these and other m a m m a l i a n and microbial sources were then tested with these antisera to determine the existence and extent of any a n t i g e n i c similarities between them. When rat and dog liver e n z y m e was allowed to diffuse against rabbit antiserum to the m a m m a l i a n urate oxidase a reaction of complete identity was obtained. This is typical of the result obtained with extracts of mammalian species (Fitzpatrick a n d McGeeney, 1975a). This is net surprising as nonm a m m a l i a n vertebrate sources (fish) also cross react with antisera to this e n z y m e although these reactions are only of partial identity (Fitzpatrick, 1972).
Fig. I--Immunological relationship between mammalian urate oxidase : Centre well--rabbit antiserum to pork liver urate oxJdase Top and bottom wells--homologous antigen Side wells--rat liver urate oxidase.
tMMUNOLOG~CALASPECTSOF URATE OX~DASETHERAPY When tested against the homologous antigen (po~k) rat and dog urate oxidase gave reactions of partial identity [Fig. 1]. However, when preparations of mic[obial urate oxidase was tested with the antiserum to pig liver enzymethey failed to cross react (F~g. 2). tn a~ a tota~ of ten microbial preparations pf urate oxidose were incubated with this antiserum but all failed to cross react with it Conversely when urate oxidase from five mammalian .sources (pig, ox. sheep, dog and rat) were studied with antisera to three of the microbial enzymes no c~oss reactions were observed indicating considerable structural differences between mammalian and microbial urate oxidase. In contrast to the close relationship observed among the mammalian urate o• {he microbial enzyme possessed a high degree of antigenic independ-
157
ence. A preparation of enzyme from a fungus A, oryzae failed to react with antiserum to B fastidiosus urate oxidase and when tested with antiserum to the A. ftavus enzyme yielded only a reaction of parPa[ identity (Fig, 3) indicating that even closely related species possess substamial differences in thei[ structure. When tested with these three antisera other microbial preparations likewise yielded a high level of independence exhibiting only limited cross reactions (Fitzpatrick and MeGeeney, 1975b). Despite the occurrence of a strong reaction between the homologous antigens and antisera as evidenced by clear and sharp precipitin lines, indicative ot high antlgenicity, inhibition of enzyme activity by homologous antiserum was not extensive (Fig. 4). This fortuitous observation confirms and extends the observation of Royer et of. (1968) that the active centre oi the molecule is not
Fig. S Non ident}ty between mammalianand micrOblal urate oxidase. Centre well--guineapig antiserum to po~k llv~t urate exldase Well 1--homologous antigen, Well~ 2, 3 and 4-A. #avus, C. ut,lis and ~. tastidiosus urale o• respectively.
F~g. 3 Immunological relationship between fungal urate ox~dase. Centre well~uinea-pig antJserur~ to A. flavus urate exidase Top and bottom wells homologous antigen. Side wells--A, oryzae urate oxidase.
,o, ~
i ~,
I
50
i00
15G
~iI A N T I S E R U M FJg. 4--Inhibition of B. fastidiosus urate oxidase by homologous antiserum. before centrifugation. * . . . . Activity 8fter centrifugation. 9Compared to control (normal serum),
*ActJvdy
IMMUNOLOGICALASPECTS OF URATE OXIDASETHERAPY in itself antigenic, nor is it sterically hindered by the presence of antibody molecules at other determinant sites. In view of the extensive immunological homology exhibited by vertebrate urate oxidase particularly, the possibility of altering cross reacting determinants by chemical modification was invesEgated. The pork enzyme-antiserum system was selected for this study. Samples of the enzyme were modified by both group specific reagents and non-specific bifunctional reagents, With the exception of glutaraldehyde all of the reagents used resulted in inactivation of the enzyme, The extent of inactivation depended upon the concentration of the reagent and the conditions employed. These pargally inactivated preparations were compared to unmodified enzyme by immunadiffusion All exhibited patferns of complete identity with the native enzyme indicating that no alteration in the determinact groups had taken place. Although treatment with glutaraldehyde did not inactivate pig liver urate oxidase it did result in polymerisation of the enzyme 1o a high molecular weight form (Fig 5). The enzyme fraction which eluted in the V~ on Sephadex G-2O0 was
Discussion The therapeutic use of enzyme in various clinical conditions, particularly those associated with simple metabolic disorders, h~s been the subiect of m~oh interest in recent years (Johnson et al., 1973; Hug, 1974; Brady etal., 1975), In particular the use of asparaginase in the treatment of lymphatic leukaemia has found wide applications. Enzyme replacement therapy is perhaps most suitable in those disorders where a build up of a catabolic end product Occurs which is no longer reuseable by the body. Urate oxidase would appear to be a suitable substance for subjects in URATE OXIDASE . . . . .
O. i 0 0 5 ~"
~.~
159
isolated, concentrated and tested with the antiserum. Polymerisation did not however result in masking of any of the determinant groups on the molecule as once again a reaction of complete identity with native pork liver urate oxidase was obtained. AS it is known that the pork enzyme is quite stable to heat (Pitts et al,, 1974) samples of the enzyme were incubated at 37'C for 15, 30 and 60 mine but no alteration in the determinant groups occur compared to an unheated control
/i ....
0.075
mL~ o.os[
,/ ,,J
," ~ ' ,
\ _
" 20 30 40 so FRACTION NUMBER Fig. 5--Chromatography of glularoldehyde lreated urate oxidase on Sephadex G-2O0
160
IRISH JOURNAL OF MEDICAL SCIENCE
whom the use of the more standard drugs (e.g. a l l o p u r i n o l , probenecid) is contraindicated e.g, severe hyperuricaemia associated with kidney nephropathy. The intravenous administration of an enzyme is not h o w e v e r without its problems as has been indicated (FitzGerald et aL, f975). In particular, the occurrence of antibodies to the e n z y m e administered c o u l d seriously reduce its efficiency in two ways. Firstly the circulating antibodies could i n h i b i t e n z y m e activity by b i n d i n g to the enzyme and blocking the active centre thereby preventing substrate m o l e c u l e s from being catalysed. Secondly, b i n d i n g of the a m l b o d y m o l e c u l e s result in accelerated removaI of the a n t i g e n (enzyme) from the eirculagon thus shortening the duration of its activity, This has already been observed in some patients being treated with urate o x i d a s e (Kissel et al,, 1968). O u r studies i n d i c a t e that the former is unlikely to o c c u r in urate oxidase therapy as we have not observed any excessive inhibition of e n z y m e activity by h o m o l o g o u s antiserum. However, the e n z y m e possesses considerable antigenicity as good a n t i b o d y titres can be readily achieved in both guinea-pigs and rabbits with both m a m m a l i a n and microbial urate oxidase, l m m u n o l o g i c a l l y the enzyme from these two sources is otherwise quite distinct as can be seen from the absence of a n y cross reactions between microbial urate oxidase and antisera to pig liver urate o x i d a s e on the o n e hand and between the m a m m a l i a n e n z y m e and antisera to microbial urate o x i d s s e on the other. However, urate o x i d a s e from closely related species m a y cross react a n d this iS particularly true of the vertebrate enzyme. T h e sensitivity of the e n z y m e to chemical modification prevents successful alteration of the determinant g r o u p s of the molecule. It would appear that the active centre of the e n z y m e is located at a position on the molecule w h i c h is not very ex-
posed. This is compatible with the relatively small size of its substrate (Molecular Weight 168} and results in the apparent absence of any determinants at or near the active centre. This is, from the point of v i e w of therapy, a fortuitous o c c u r r e n c e as the production of antibodies to determinant g r o u p s associated with the active centre would almost certainly result not only in inhibition of enzyme activity but atso in a m u c h h i g h e r level of cross reaction. The m e c h a n i s m s of enzymatic o x i d a l i o n of urate is identical for both pig river and A. flavus enzyme ( H u b s c h e r et al,, 1957; Labourer and Langlois, t968) suggesting little variation in a m i n o acid sequences involved in catalysis. T h e p r o c e d u r e evolved for this study on urate o x i d a s e w o u l d have a p p l i c a t i o n to s i m i l a r investigations on other e n z y m e s suggested for replacement therapy. D. A. F. acknowledges feeelpl of a Fellowship of the Medica~ Research Council of Irelaqd References Brady, R O, Dentcher, P G and Gal, A E 1975. Inves#galions in enzyme replacement therapy in lipid storage diseases. Fed. Proc 34, 1310. Brogard, J. M., Coumaros, D. FranckHauser, J. Stahl, A. and Stahl, J 1972. Enzymatic Uricotysis: A study of the effect of a fungal urate oxidase. Europ J. Clin. Biol. Res. 17. a90 FitzGerald, O., Fitzpatrick, D. A. and McGeeney K. F. 1975. Urate oxidase treatmert for hyper~ uricaemia Lancet i, 525. Fitzpatrick, D. A. 1972. Ph.D Thesis National University of Ireland. Filzpatrick, B A. and McGeeney, K F 1975a Comparative immunology of verlebrate urate oxidase. Comp. Bioohem Physiol. 51B. 37, Filzpatrick, D. A. and McGeeney, K F 1975b, Antigenic independence of some microbial tlrate oxidase. Infect. Immun. 12, 1237. Graham, R. C and K~rtlovsky, M. J. ~965 The histeohemica[ demonstration of uricase acSvily. J Hsochem Oyochem. 13, 448 Hubscher G.. 8aura, H and Mahler, H R 1957. Studies on Udcase IV. The nature and composition of some stable reaction products, B[ochem. biophys. Acts 23, 43 Hug, G. 1974. Enzyme therapy and 0renatal diagnosis in glycogenos[s Type II Am J Dis. Child. 128, 607.
Johnson, ~v', G., Desniok, R. J. and Long, D.M. 1973, Intravenous injection of purified hexosaminidase A ~nto a patient with Tay-$achs disease. Birth Defects 9, 120. KJssel, P, Lamarche, M, and Rayer, R. 1968. Modification of uricaemia and the excretion of uric acid nffrogen by an enzyme of fungal origin Nature (London) 217, 72. Labourer, P. and Langlois, C. 1968 Urate o• dase d'Aspergillus flavus. I1. Metabolisme inhibitions, specifieite. Bull. Soc. Chim, blol 50, 827. LondQn, NI and Hudson. P. B 1957 Urlcolytic activily of purified uricase in Iwe human beJags, Science N.Y. 125, 937. Means, G. E. aud Feeney, R. F. 197~. Chemical modificaIfon of proteins, San Frenoisco HoF den-Day Inc.
Pdts, O, M., Palest, O. G. and Fish, W. W. 1974 Uricase Subunit composition and resistance to denaturants. Biochemistry 13, 888. Royer, R., Vinde], J., Lamarehe, M. and K~ssel, P. 1968. Medaldies of purine excretion during enzyme treatmen~ of gout and other hypercuricomic conditions w~th urate exidase. Presse Med. 76, 2325. Snyder, P, D., Wold, F., Bernlohr, R. W., Dullam, C., Oesnick, R. J , Krivit, W. and Condie, R. M. 1974. Enzyme Therapy II, Purified human and ga~actosidase A Biochim. Biophys. Acta 350, 432. Zittoun, R, Dauchy, F, Teirlaud, C., Barth~lemy, M. and Bouchard, P. 1976. Le traJtment des hyperuricem~es en hemato}og~e par I'urateoxydase et I'~l[opurinol Ann, M6d. interne ~72, 6 7, 479.
body production is a function of the individual antigen used. As the immunelegy of urate oxidase has not previously been the subject of extensive study we felt it would be of interest to study the antigenic properties of this enzyme so that its clinical use can take place against a competent knowledge of the potential immunological problems which might arise. In addition the possibility of decreasing the extent of cross reaction by chemical modification of the enzyme was investigated.
Materials and Methods Antisera were produced by administering 5.0 I.U. (1 ml) of enzyme emulsified with an equal volume of Freund's complete adjuvant subcutaneously. Animals were bled after three or more injections. Pork and rat liver urate oxidase were purified by a procedure reported elsewhere (Fitzpatrick, 1972). Aspergillu~ oryzae (Botany Department, U.C.D., No. 355) was grown on minimal medium containing 0.05 per cent urate and the enzyme extracted and partially purified by ammonium sulphate fractionation. Candida utilis urate oxidase was Type IV from Sigma. L. Mahler donated the Bacillus fastidiosus enzyme and P. Labcurer the Aspergillus flavus enzyme, Ouchterlony immunodiffusion was carried out on 1 per cent agarose (Difco) plates. These were stained specifically for urate oxidase activity (Graham and Karnovsky, 1965). Enzyme inhlbi6on was studied by incubating samples of B, fastidiosus urate oxidase (0.5 ml, 0.28 I.U.) at room temperature with increas~ ing amounts of homologous antiserum for 10 minutes and assayed for enzyme
155
156
IRISH JOURNAL OF MEDICAL SCIENCE
activity. After s t a n d i n g overnight the solutions were centrifuged and the supernatants assayed for activity. All e n z y m e modifications were carried out on the presence of 0.5 per cent bovine serum albumin. Polymerisation with glu{araldehyde (0.0015 per cent) was carried out at pH 7, all other reactions at pH 10.0 in 0.1 M sodium carbonate. Treatment with h e x a m e t h y l e n e diisocyanate was as described by Synder et al. (1974), other reaction c o n d i t i o n s were those of Means and Feeney (1971). T h e modifying reagents used were, amino g r o u p s : acetic a n d s u c c i n i c anhydride, ethylacetimidate and S-ethylthioltrifluoroacetate. G u a n i d i n o groups : cycIohexanedione and phenylglyoxal. Phenolic g r o u p s : tetranitromethane. Imdazole g r o u p s : diethylpyrocarbonate. Bifunctional reagents : h e x a m e t h y l e n e diisocyanate a n d glutaraidehyde.
Results Antisera were prepared to purified preparations of ura~e oxidase from a m a m m a l i a n (pig liver) and three microbia[ species (C. ufilis, A. flavus and B. fastidiosus). Urate oxidase from these and other m a m m a l i a n and microbial sources were then tested with these antisera to determine the existence and extent of any a n t i g e n i c similarities between them. When rat and dog liver e n z y m e was allowed to diffuse against rabbit antiserum to the m a m m a l i a n urate oxidase a reaction of complete identity was obtained. This is typical of the result obtained with extracts of mammalian species (Fitzpatrick a n d McGeeney, 1975a). This is net surprising as nonm a m m a l i a n vertebrate sources (fish) also cross react with antisera to this e n z y m e although these reactions are only of partial identity (Fitzpatrick, 1972).
Fig. I--Immunological relationship between mammalian urate oxidase : Centre well--rabbit antiserum to pork liver urate oxJdase Top and bottom wells--homologous antigen Side wells--rat liver urate oxidase.
tMMUNOLOG~CALASPECTSOF URATE OX~DASETHERAPY When tested against the homologous antigen (po~k) rat and dog urate oxidase gave reactions of partial identity [Fig. 1]. However, when preparations of mic[obial urate oxidase was tested with the antiserum to pig liver enzymethey failed to cross react (F~g. 2). tn a~ a tota~ of ten microbial preparations pf urate oxidose were incubated with this antiserum but all failed to cross react with it Conversely when urate oxidase from five mammalian .sources (pig, ox. sheep, dog and rat) were studied with antisera to three of the microbial enzymes no c~oss reactions were observed indicating considerable structural differences between mammalian and microbial urate oxidase. In contrast to the close relationship observed among the mammalian urate o• {he microbial enzyme possessed a high degree of antigenic independ-
157
ence. A preparation of enzyme from a fungus A, oryzae failed to react with antiserum to B fastidiosus urate oxidase and when tested with antiserum to the A. ftavus enzyme yielded only a reaction of parPa[ identity (Fig, 3) indicating that even closely related species possess substamial differences in thei[ structure. When tested with these three antisera other microbial preparations likewise yielded a high level of independence exhibiting only limited cross reactions (Fitzpatrick and MeGeeney, 1975b). Despite the occurrence of a strong reaction between the homologous antigens and antisera as evidenced by clear and sharp precipitin lines, indicative ot high antlgenicity, inhibition of enzyme activity by homologous antiserum was not extensive (Fig. 4). This fortuitous observation confirms and extends the observation of Royer et of. (1968) that the active centre oi the molecule is not
Fig. S Non ident}ty between mammalianand micrOblal urate oxidase. Centre well--guineapig antiserum to po~k llv~t urate exldase Well 1--homologous antigen, Well~ 2, 3 and 4-A. #avus, C. ut,lis and ~. tastidiosus urale o• respectively.
F~g. 3 Immunological relationship between fungal urate ox~dase. Centre well~uinea-pig antJserur~ to A. flavus urate exidase Top and bottom wells homologous antigen. Side wells--A, oryzae urate oxidase.
,o, ~
i ~,
I
50
i00
15G
~iI A N T I S E R U M FJg. 4--Inhibition of B. fastidiosus urate oxidase by homologous antiserum. before centrifugation. * . . . . Activity 8fter centrifugation. 9Compared to control (normal serum),
*ActJvdy
IMMUNOLOGICALASPECTS OF URATE OXIDASETHERAPY in itself antigenic, nor is it sterically hindered by the presence of antibody molecules at other determinant sites. In view of the extensive immunological homology exhibited by vertebrate urate oxidase particularly, the possibility of altering cross reacting determinants by chemical modification was invesEgated. The pork enzyme-antiserum system was selected for this study. Samples of the enzyme were modified by both group specific reagents and non-specific bifunctional reagents, With the exception of glutaraldehyde all of the reagents used resulted in inactivation of the enzyme, The extent of inactivation depended upon the concentration of the reagent and the conditions employed. These pargally inactivated preparations were compared to unmodified enzyme by immunadiffusion All exhibited patferns of complete identity with the native enzyme indicating that no alteration in the determinact groups had taken place. Although treatment with glutaraldehyde did not inactivate pig liver urate oxidase it did result in polymerisation of the enzyme 1o a high molecular weight form (Fig 5). The enzyme fraction which eluted in the V~ on Sephadex G-2O0 was
Discussion The therapeutic use of enzyme in various clinical conditions, particularly those associated with simple metabolic disorders, h~s been the subiect of m~oh interest in recent years (Johnson et al., 1973; Hug, 1974; Brady etal., 1975), In particular the use of asparaginase in the treatment of lymphatic leukaemia has found wide applications. Enzyme replacement therapy is perhaps most suitable in those disorders where a build up of a catabolic end product Occurs which is no longer reuseable by the body. Urate oxidase would appear to be a suitable substance for subjects in URATE OXIDASE . . . . .
O. i 0 0 5 ~"
~.~
159
isolated, concentrated and tested with the antiserum. Polymerisation did not however result in masking of any of the determinant groups on the molecule as once again a reaction of complete identity with native pork liver urate oxidase was obtained. AS it is known that the pork enzyme is quite stable to heat (Pitts et al,, 1974) samples of the enzyme were incubated at 37'C for 15, 30 and 60 mine but no alteration in the determinant groups occur compared to an unheated control
/i ....
0.075
mL~ o.os[
,/ ,,J
," ~ ' ,
\ _
" 20 30 40 so FRACTION NUMBER Fig. 5--Chromatography of glularoldehyde lreated urate oxidase on Sephadex G-2O0
160
IRISH JOURNAL OF MEDICAL SCIENCE
whom the use of the more standard drugs (e.g. a l l o p u r i n o l , probenecid) is contraindicated e.g, severe hyperuricaemia associated with kidney nephropathy. The intravenous administration of an enzyme is not h o w e v e r without its problems as has been indicated (FitzGerald et aL, f975). In particular, the occurrence of antibodies to the e n z y m e administered c o u l d seriously reduce its efficiency in two ways. Firstly the circulating antibodies could i n h i b i t e n z y m e activity by b i n d i n g to the enzyme and blocking the active centre thereby preventing substrate m o l e c u l e s from being catalysed. Secondly, b i n d i n g of the a m l b o d y m o l e c u l e s result in accelerated removaI of the a n t i g e n (enzyme) from the eirculagon thus shortening the duration of its activity, This has already been observed in some patients being treated with urate o x i d a s e (Kissel et al,, 1968). O u r studies i n d i c a t e that the former is unlikely to o c c u r in urate oxidase therapy as we have not observed any excessive inhibition of e n z y m e activity by h o m o l o g o u s antiserum. However, the e n z y m e possesses considerable antigenicity as good a n t i b o d y titres can be readily achieved in both guinea-pigs and rabbits with both m a m m a l i a n and microbial urate oxidase, l m m u n o l o g i c a l l y the enzyme from these two sources is otherwise quite distinct as can be seen from the absence of a n y cross reactions between microbial urate oxidase and antisera to pig liver urate o x i d a s e on the o n e hand and between the m a m m a l i a n e n z y m e and antisera to microbial urate o x i d s s e on the other. However, urate o x i d a s e from closely related species m a y cross react a n d this iS particularly true of the vertebrate enzyme. T h e sensitivity of the e n z y m e to chemical modification prevents successful alteration of the determinant g r o u p s of the molecule. It would appear that the active centre of the e n z y m e is located at a position on the molecule w h i c h is not very ex-
posed. This is compatible with the relatively small size of its substrate (Molecular Weight 168} and results in the apparent absence of any determinants at or near the active centre. This is, from the point of v i e w of therapy, a fortuitous o c c u r r e n c e as the production of antibodies to determinant g r o u p s associated with the active centre would almost certainly result not only in inhibition of enzyme activity but atso in a m u c h h i g h e r level of cross reaction. The m e c h a n i s m s of enzymatic o x i d a l i o n of urate is identical for both pig river and A. flavus enzyme ( H u b s c h e r et al,, 1957; Labourer and Langlois, t968) suggesting little variation in a m i n o acid sequences involved in catalysis. T h e p r o c e d u r e evolved for this study on urate o x i d a s e w o u l d have a p p l i c a t i o n to s i m i l a r investigations on other e n z y m e s suggested for replacement therapy. D. A. F. acknowledges feeelpl of a Fellowship of the Medica~ Research Council of Irelaqd References Brady, R O, Dentcher, P G and Gal, A E 1975. Inves#galions in enzyme replacement therapy in lipid storage diseases. Fed. Proc 34, 1310. Brogard, J. M., Coumaros, D. FranckHauser, J. Stahl, A. and Stahl, J 1972. Enzymatic Uricotysis: A study of the effect of a fungal urate oxidase. Europ J. Clin. Biol. Res. 17. a90 FitzGerald, O., Fitzpatrick, D. A. and McGeeney K. F. 1975. Urate oxidase treatmert for hyper~ uricaemia Lancet i, 525. Fitzpatrick, D. A. 1972. Ph.D Thesis National University of Ireland. Filzpatrick, B A. and McGeeney, K F 1975a Comparative immunology of verlebrate urate oxidase. Comp. Bioohem Physiol. 51B. 37, Filzpatrick, D. A. and McGeeney, K F 1975b, Antigenic independence of some microbial tlrate oxidase. Infect. Immun. 12, 1237. Graham, R. C and K~rtlovsky, M. J. ~965 The histeohemica[ demonstration of uricase acSvily. J Hsochem Oyochem. 13, 448 Hubscher G.. 8aura, H and Mahler, H R 1957. Studies on Udcase IV. The nature and composition of some stable reaction products, B[ochem. biophys. Acts 23, 43 Hug, G. 1974. Enzyme therapy and 0renatal diagnosis in glycogenos[s Type II Am J Dis. Child. 128, 607.
Johnson, ~v', G., Desniok, R. J. and Long, D.M. 1973, Intravenous injection of purified hexosaminidase A ~nto a patient with Tay-$achs disease. Birth Defects 9, 120. KJssel, P, Lamarche, M, and Rayer, R. 1968. Modification of uricaemia and the excretion of uric acid nffrogen by an enzyme of fungal origin Nature (London) 217, 72. Labourer, P. and Langlois, C. 1968 Urate o• dase d'Aspergillus flavus. I1. Metabolisme inhibitions, specifieite. Bull. Soc. Chim, blol 50, 827. LondQn, NI and Hudson. P. B 1957 Urlcolytic activily of purified uricase in Iwe human beJags, Science N.Y. 125, 937. Means, G. E. aud Feeney, R. F. 197~. Chemical modificaIfon of proteins, San Frenoisco HoF den-Day Inc.
Pdts, O, M., Palest, O. G. and Fish, W. W. 1974 Uricase Subunit composition and resistance to denaturants. Biochemistry 13, 888. Royer, R., Vinde], J., Lamarehe, M. and K~ssel, P. 1968. Medaldies of purine excretion during enzyme treatmen~ of gout and other hypercuricomic conditions w~th urate exidase. Presse Med. 76, 2325. Snyder, P, D., Wold, F., Bernlohr, R. W., Dullam, C., Oesnick, R. J , Krivit, W. and Condie, R. M. 1974. Enzyme Therapy II, Purified human and ga~actosidase A Biochim. Biophys. Acta 350, 432. Zittoun, R, Dauchy, F, Teirlaud, C., Barth~lemy, M. and Bouchard, P. 1976. Le traJtment des hyperuricem~es en hemato}og~e par I'urateoxydase et I'~l[opurinol Ann, M6d. interne ~72, 6 7, 479.
