Novel multi-component conjugates are provided having at least two components which are superoxide dismutase and catalase. Novel therapeutic agents utilising these conjugates are provided which are effective to reduce harmful levels of superoxide and hydroxyl free radicals in body tissues.

TECHNICAL FIELD 
This invention relates to novel superoxide dismutase-catalase conjugates 
and to novel therapeutic agents utilising said conjugates. 
BACKGROUND OF THE INVENTION 
The human and animal body produces highly reactive free radicals by a 
variety of normal metabolic processes. The action of xanthine oxidase on 
xanthine, for example, results in the single-electron reduction of oxygen 
and produces both superoxide and hydroxyl free radicals (Kuppusamy & 
Zweier (1989) J. Biol. Chem. 264, 9880-9884). Free radicals have been 
implicated in the causation of a wide range of clinical conditions 
including atherosclerosis, rheumatoid arthritis, cancer, pulmonary 
diseases of the newborn and the normal ageing process, and have been 
reported to be associated with reperfusion injuries following ischemic 
episodes associated with myocardial infarct, cerebral ischemia and 
vasospasm and surgical intervention. 
Superoxide and hydroxyl free radicals and hydrogen peroxide can result in 
peroxidation of membrane phospholipids and oxidation of cellular proteins 
and nucleic acids. These species are thought to be involved in various 
pathological conditions including tissue injury, inflammatory conditions 
and radiation damage. 
A variety of endogenous defence mechanisms are thought to protect the 
organism from the deleterious effects of these reactive oxygen species 
under normal physiological conditions. One of these is the enzyme, 
superoxide dismutase (SOD), which occurs widely in prokaryotes and 
eukaryotes. SOD catalyses the dismutation of the superoxide free radical 
(O.sub.2.), in the presence of hydrogen, to produce hydrogen peroxide. 
Hydrogen peroxide, which is itself a potentially deleterious reactive 
species, is reduced to oxygen and water by the enzyme, catalase. Catalase 
also destroys the hydroxyl free radical (OH.) by removing H.sub.2 O.sub.2 
and hence decreasing the reaction of H.sub.2 O.sub.2 with O.sub..sub.2. to 
produce OH. 
The effect of exogenously administered SOD as a therapeutic agent to 
protect against superoxide free radical damage has been studied in mammals 
with mixed results. The variability of the effect produced by SOD makes it 
somewhat unsatisfactory as a therapeutic agent. 
One factor contributing to the variability of effect found with exogenous 
SOD administration is the very short half-life of SOD in the mammalian 
circulation, of the order of 4 to 5 minutes. 
Previous work has shown that the half-life of SOD in the circulation can be 
increased by conjugation of the enzyme with a larger molecule, for 
example, albumin or polyethylene glycol (PEG). SOD-albumin conjugates have 
circulation half-lives of 4-6 hours and show reduced immunogenicity 
compared to SOD alone (Mao & Poznansky (1989), Biomat. Art. Cells & Art. 
Org., v. 17 (3) , p. 229-244). 
Another factor which likely plays a role in the variable results found with 
exogenous SOD administration is the strong inhibition of SOD by hydrogen 
peroxide, one of the products of its own catalytic activity. 
A further limitation on the usefulness of SOD, and also of SOD-albumin 
conjugates, is that SOD is not able to remove hydroxyl free radicals. At 
high levels of exogenous SOD administration, SOD actually increases 
hydroxyl free radical production and a similar effect is seen with high 
levels of SOD-albumin conjugate. 
The SOD-careless conjugates of the invention are advantageous as novel free 
radical scavengers capable of removing both oxygen and hydroxyl free 
radicals. 
DISCLOSURE OF THE INVENTION 
According to one aspect of the invention, a multi-component conjugate 
having at least two components is provided, wherein said at least two 
components are superoxide dismutase and catalase. 
According to a further aspect of the invention, a pharmaceutical 
composition is provided comprising a multi-component conjugate having at 
least two components, wherein the at least two components are superoxide 
dismutase and catalase, in an amount effective to reduce harmful levels of 
superoxide and hydroxyl free radicals in body tissues. 
According to a further aspect of the invention, a method of treatment of a 
subject having a condition in which superoxide and hydroxyl free radicals 
play a harmful role is provided, said method comprising administration to 
said subject, in an amount effective to reduce the tissue levels of said 
free radicals, of a multi-component conjugate having at least two 
components wherein the at least two components are superoxide dismutase 
and catalase in a pharmaceutically acceptable carrier. 
According to a further aspect of the invention, a method of treatment of a 
subject is provided to reduce tissue levels of superoxide and hydroxyl 
free radicals in said subject, said method comprising administration to 
said subject of an effective amount of a conjugate of superoxide diemurals 
and catalase in a pharmaceutically acceptable carrier. 
The invention, as exemplified by preferred embodiments, is described with 
reference to the drawings.

MODES OF CARRYING OUT THE INVENTION 
SOD and catalase are "conjugated", as that term is used herein, when they 
are attached to each other in such a manner as to resist separation under 
normal physiological conditions. It is presently preferred to conjugate 
SOD and catalase chemically, using a divalent linking group such as 
glutaraldehyde or carbodiimide. However, one may employ any suitable 
linking group which does not result in loss of enzyme activity to an 
unacceptable degree. A number of linking groups are commonly used for 
conjugating antibodies to enzymes used for signal amplification in ELISA 
methods, and are generally suitable for the practice of the present 
invention. Alternatively, one may conjugate SOD and catalase indirectly, 
by conjugating each one, individually or simultaneously, to a 
macromolecular carrier such as albumin (preferably HSA), polyethylene 
glycol, starch, and the like. In general, any soluble nonimmunogenic 
macromolecule should be acceptable as a carrier for conjugates of the 
invention. 
Conjugation need not be covalent: for example, one may conjugate catalase 
and SOD to individual binding partners which bind by non-covalent 
interactions. For example, one may conjugate SOD to avidin and conjugate 
catalase to biotin: conjugates of the invention are then prepared by 
mixing SOD-avidin and catalase-biotin, and allowing the avidin-biotin 
affinity to effectively couple SOD and catalase. Other binding partners 
include receptor-ligand and antibody-antigen pairs, although the 
avidin-biotin pair is generally the easiest to employ and is the least 
antigenic. 
As used herein, "SOD" refers to superoxide dismutase obtained from any 
organism, and enzymes exhibiting substantially the same activity. "SOD" 
also refers to variations on the native enzyme obtained by mutagenesis, 
recombinant engineering, or other synthetic techniques. SOD derived from a 
variety of sources is commercially available. Common sources include 
bovine kidney, bovine liver, canine erythrocyte, horseradish, human 
erythrocyte, yeast, and bacteria. Similarly, "catalase" refers to any 
catalase enzyme, obtained from any organism, and variations thereof 
substantially retaining native catalase activity. Catalase derived from a 
variety of sources is also commercially available. 
"SOD-catalase conjugates" as that term is used herein, may contain 
components in addition to SOD and catalase, for example albumin or a 
targeting agent, as described further herein. 
General Method 
SOD and catalase obtained from commercial sources are suitable 
for-conjugation. Chemical conjugation may be effected by agents such as 
glutaraldehyde, which links primary amino groups, and water soluble 
carbodiimide, which links amino groups to carboxyl groups. 
By varying the conjugation conditions, SOD-catalase conjugates of different 
molecular weights and mole ratios may be prepared. 
SOD-catalase conjugates of molecular weights ranging from around 200,000 to 
around 1,200,000 have been prepared and all were active in scavenging 
superoxide and hydroxyl free radicals. Conjugates of SOD:catalase mole 
ratios ranging from 1:1 to 8:1 showed similar free radical scavenging 
ability. Various methods of conjugating or cross-linking enzymes are 
described in Poznansky, (1988) in Methods in Enzymology; Ed. K. Mosbach, 
V. 137, Immobilized Enzymes & Cells, Part D, p. 566. 
Genetic engineering techniques may also be used to effect linkage of SOD 
and catalase to provide cloned quantities of homogeneous conjugates (Bulow 
& Mosbach, Ann. N. Y. Acad. Sci., 1987, Vol. 501, p.44). Other methods of 
conjugating SOD and catalase will be known to those skilled in the art. 
The SOD-catalase conjugates of the invention retain substantially all of 
the catalase and SOD catalytic activities of the unconjugated enzymes. 
Catalase activity may be modestly decreased whereas SOD activity is 
unaffected or, where glutaraldehyde is used as conjugating agent, somewhat 
enhanced. 
The preservation of enzyme activity on conjugation is shown in Table 1. 
TABLE 1 
______________________________________ 
SOD and Catalase Activity Following Cross Linking 
with either Glutaraldehyde or Carbodiimide 
Enzyme Enzyme 
Activity Prep. % Initial Activity* 
______________________________________ 
SOD (SOD-Cat).sup.1 
110% 
Catalase (SOD-Cat).sup.1 
80% 
SOD (SOD-Cat).sup.2 
100% 
Catalase (SOD-Cat).sup.2 
72% 
______________________________________ 
*Enzyme Activity Expressed as a percentage of preconjugation activity. 
.sup.1 Conjugation by glutaraldehyde as in Example 1 
.sup.2 Conjugation by carbodiimide as in Example 3 
SOD activity before and after conjugation was assayed by the method of 
McCord & Fridovich (1969) J. Biol. Chem., v. 244, p. 6049 and catalase by 
the method of Claiborne, A. in Handbook of Methods for Oxygen Radical 
Research (1985) Ed. R. A. Greenwald, CRC Press, p. 283. 
The SOD-catalase conjugates of the invention provide novel agents which can 
destroy both superoxide and hydroxyl free radicals, both of which are 
cytotoxic. Additionally, the conjugates of the invention provide catalase 
activity adjacent to the site of H.sub.2 O.sub.2 production by the action 
of SOD, thus removing H.sub.2 O.sub.2 before it can inhibit the SOD or 
cause peroxidation of cell components. FIG. 1 shows the protective effect 
of conjugation to catalase on the inhibition of SOD by H.sub.2 O.sub.2. 
Without limiting ourselves to this one theory, one possible mechanism by 
which the catalase enzyme reduces inhibition of the SOD enzyme is 
illustrated by the following three equations. 
______________________________________ 
1. Role of SOD in dismutating O.sub.2.sup.. : 
O.sub.2.sup.. + O.sub.2.sup.. + 2H.sup.+ SOD H.sub.2 O.sub.2 + 
O.sub.2 ; 
2. Role of H.sub.2 O.sub.2 in producing .sub.----OH.: 
H.sub.2 O.sub.2 + O.sub.2.sup.. OH. + OH.sup.- + O.sub.2 ; and 
3. Role of Catalase in getting rid of H.sub.2 O.sub.2 and hence 
.sub.----OH.: 
2H.sub.2 O.sub.2 catalase 2H.sub.2 O + O.sub.2 
______________________________________ 
Other possible mechanisms and theories would be appreciated by those 
skilled in the art. 
Conjugation of SOD with catalase provides an SOD preparation having an 
extended circulation half-life suitable for therapeutic use. As measured 
in anaesthetized rate, free SOD has a half-life in the circulation of 4-5 
minutes. The SOD-catalase conjugate can remain circulating with a 
half-life of as much as 5-6 hours dependent on the molecular weight of the 
conjugate. 
Conjugates of SOD, catalase and albumin may be prepared by addition of 
albumin, preferably human serum albumin, to the pre-conjugation enzyme 
mixture, giving conjugates of reduced immunogenicity. An 
SOD-careless-albumin conjugate, prepared as described in Poznansky 
(Methods in Enzymology, supra), had a molecular weight greater than 
600,000 and retained full SOD and catalase activity. 
Targeting agents such as antibodies capable of tissue-specific or cell 
component-specific binding may also be conjugated with SOD and catalase to 
direct the conjugate to a desired site of action. 
A conjugate including SOD, catalase and a monoclonal antibody to heavy 
chain myosin was prepared as described by Poznansky (Methods in 
Enzymology, supra). This conjugate showed no loss of SOD or catalase 
activity and retained the ability of the antibody to bind to its antigen. 
The SOD-careless conjugates of the invention effectively remove superoxide 
and hydroxyl free radicals in the presence of agents such as iron which 
stimulate production of hydroxyl free radicals. If SOD alone is used in 
this situation, it has little scavenging effect on hydroxyl free radicals 
and under some circumstances itself stimulates further production of 
hydroxyl free radicals. 
Due in part to their longer circulation half-life and to the proximity of 
the catalase portion to the site of H.sub.2 O.sub.2 production by SOD, the 
SOD-careless conjugates of the invention are superior to equivalent 
amounts of unconjugated SOD and catalase in scavenging superoxide and 
hydroxyl free radicals, as seen in Example 8. 
The conjugates of the invention provide useful therapeutic agents for 
protecting organisms against damage due to superoxide and/or hydroxyl free 
radicals. In clinical situations involving a period of ischaemia followed 
by re-perfusion and re-oxygenation, reperfusion injury is common and free 
radicals have been implicated in such injury. The conjugates of the 
invention preserve cardiac function in mammalian hearts during 
re-perfusion following an ischaemic episode whereas unconjugated SOD under 
similar conditions shows little or no protective effect, as seen in 
Example 9. The conjugates of the invention provide novel therapeutic 
agents useful in such clinical situations to inactivate both superoxide 
and hydroxy free radicals. It will be appreciated by one skilled in the 
art that an SOD-catalase conjugate could be applied in numerous other 
clinical situations in which the removal of free radicals is desired. 
The extended half-life of the conjugates of the invention in the 
circulation provides a superior therapeutic agent to the quickly-excluded 
free SOD. 
The conjugates of the invention provide therapeutic agents useful in 
clinical conditions in which damage is caused by superoxide and hydroxyl 
free radicals or in clinical situations in which H.sub.2 O.sub.2 is 
produced, including situations in which SOD is desirably employed to 
inactivate superoxide free radicals. 
Further details of the preferred embodiments of the invention will be 
understood from the following examples which are understood to be 
non-limiting. 
EXAMPLES 
Example 1 
Conjugation by glutaraldehyde 
5 mg. SOD (yeast SOD purified to homogeneity (99% by SDS Page), specific 
activity 5500 U/mg, Sigma) and 2 mg. catalase (porcine liver, specific 
activity 10,000 U/mg) were dissolved in phosphate-buffered saline, pH 7.4. 
100 .mu.l of glutaraldehyde (25%) was added with stirring at 4.degree. C. 
for 3h. The reaction was stopped by the addition of 50 mg of glycine, the 
solution was dialyzed overnight against phosphate buffered saline and the 
product was sized and purified by molecular sieve chromatography using 
Sephadex G-150 (trade-mark). 
On chromatography of the product on Sephadex G-150, 90% of the SOD activity 
eluted between molecular weights 250,000 and 650,000 with peak activity at 
450,000. Profiles of catalase activity and protein content of the eluted 
fractions were almost identical to the SOD profile. 
Rechromatography of the peak fractions on Sephadex G-150 gave a product of 
molecular weight 450,000.+-.50,000 (mean.+-.1 Standard Deviation). 
Recovery of enzyme activity was 110% for SOD and 80% for catalase. Mole 
ratio of SOD:catalase was 4. 
Example 2 
Conjugation by Glutaraldehyde 
2mg SOD plus 1 mg catalase (activities as in Example 1) were dissolved in 4 
ml of phosphate buffered saline (PBS, pH7.4) to which 50 .mu.l of 
glutaraldehyde was added with stirring for 1h at 4.degree. C. The reaction 
was stopped as in Example 1 and purification and assay of the conjugate 
was carried out. These conditions yielded a conjugate with a smaller 
molecular weight (250,000.+-.40,000) with a mole ratio of 8 (SOD/catalase) 
and with enzyme activity recoveries of 100% and 75% for the SOD and 
catalase respectively. 
SOD-careless conjugates of different molecular weights or SOD:catalase mole 
ratios may be prepared by varying the conditions of the cross-linking 
reaction in terms of the starting mole ratio of unconjugated enzymes, 
concentration of cross-linking agent and reaction time and temperature, as 
is known to one skilled in the art. 
Example 3 
Conjugation by carbodiimide 
20 .mu.g water soluble carbodiimide EDCI 
(1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide HCl) was added with 
stirring to 2mg SOD dissolved in PBS at 4.degree. C. for 8 hours. 
The reaction mixture was dialysed against PBS for a minimum of 4 hours to 
remove unreacted carbodiimide and catalase (0.5 mg) was added, with 
continued stirring at 4.degree. C. for 2 hours. Reaction was stopped as in 
Example 1 and the product was sized and purified as in Example 1. 
The purified product had a molecular weight of 600,000 and a mole ratio 
SOD:catalase of 3. 
Example 4 
Half-life of SOD-catalase Conjugates 
SOD-catalase conjugates were radiolabelled by standard methods and their 
half-lives were determined in rate anaesthetised with Nembutal. 
The radiolabelled conjugate (1 million counts) was injected intravenously 
with or without a 100 fold excess of unlabelled conjugate. Blood samples 
were drawn every 30 minutes. The conjugate prepared as in Example 1 had a 
circulation half-life of 300 minutes compared to the half-life of 5 
minutes for the native or unmodified SOD and 120 minutes for the 
unmodified catalase. 
Conjugates examined had half-lives ranging from 60 minutes to 420 minutes 
depending on the SOD : catalase mole ratio and the molecular weight of the 
conjugate. 
Example 5 
Scavenging of Free Radicals by SOD-catalase Conjugates 
SOD-catalase conjugate was prepared as in Example 1. 
Xanthine and xanthine oxidass were used to generate superoxide free 
radicals and hydroxyl free radicals, using the system of Fridovich, I., 
Xanthine Oxidass in: Handbook of Methods for Oxygen Radical Research 
(1985) Ed. R. A. Greenwald, CRC Press, p. 51. 
Free radicals were detected by Electron paramagnetic Resonance (EPR) spin 
trapping with DMPO as described by Thornalley, P. J. & Bannister, J. V., 
The Spin Trapping of Superoxide Radicals in Handbook of Methods for Oxygen 
Radical Research (1985) Ed. R. A. Greenwald, CRC Press, p. 133. 
The xanthine (X) plus xanthine oxidass (XO) reaction was allowed to proceed 
at 37.degree. C. in the presence or absence of free catalase (Cat), free 
SOD or SOD-catalase conjugates (SOD-Cat). At t=0 or t=10 min, the spin 
trapping agent DMPO was added and the sample was placed in a Bruker 300 
EPO spectrometer. 
EPR peaks were scored on an intensity scale of 0-10 and the results are set 
out in Table 2 
TABLE 2 
______________________________________ 
SOD, Catalase and SOD-catalase Mediated Scavenging 
of Superoxide (O.sub.2.sup.. ) and Hydroxyl (OH.) Free Radicals 
O.sub.2.sup.. 
OH 
______________________________________ 
X + XO t = 1 min 8 1 
X + XO t = 10 min 10 4 
X + XO + SOD t = 1 min 0 1 
X + XO + SOD t = 10 min 0 2 
X + XO + SOD - Cat 
t = 1 min 0 0 
X + XO + SOD - Cat 
t = 10 min 0 0 
X + XO + Cat t = 1 min 8 0 
X + XO + Cat t = 10 min 10 2 
______________________________________ 
Example 6 
SOD-catalase conjugate was prepared as in Example 1. 
Xanthine and xanthine oxidass were used to generate superoxide free 
radicals and hydroxyl free radicals as in Example 5. 
Replicate samples were treated as shown in the panels of FIG. 2 and 
resultant free radical levels were measured by EPR as described in Example 
5. FIG. 2 shows the results of this experiment. 
Panel A shows generation of superoxide free radicals and hydroxyl free 
radicals in the absence of further additions to the reaction mixture. 
Addition of SOD gave scavenging of virtually all oxygen free radicals but 
left some hydroxyl free radical unscavenged (Panel B). Addition of 0.1 mM 
FeCl.sub.3 greatly diminished the O. signal while stimulating the OH. 
signal (Panel C). 
Addition of SOD along with 0.1 mM FeCl.sub.3 (for up to 2 h) did little to 
remove the increased OH. radical (Panel D) whereas addition of 
SOD-catalase conjugate virtually eliminated the iron-stimulated OH. 
radical. 
Increasing doses of SOD had no effect on the size of the OH. signal except 
at higher iron concentrations where the OH. signal was then increased with 
increasing SOD due to the iron and the generation of H.sub.2 O.sub.2. 
Example 7 
The experiment of Example 6 was repeated using 0.5 mM FeCl.sub.3 
+SOD-catalase conjugate prepared as in Example 1, and the results are 
shown in FIG. 3. As seen in Panel A, in the presence of 0.5 mM FeCl.sub.3 
only OH. was readily discerned. The iron assured conversion of the O. to 
OH. via the Haber-Weiss reaction. The addition of SOD to the reaction 
further stimulated OH. production (Panel B) presumably by the action of 
H.sub.2 O.sub.2. This indicates the potential toxicity of SOD alone, in 
that it actually generates the more toxic OH free radical species. Panels 
C and D show that addition of SOD-catalase conjugates, (500 Units and 2000 
Units (high) SOD respectively) completely removed OH. radical even in the 
presence of 0.5 mM FeCl.sub.3. 
Example 8 
The experimental set up of Example 7 was used to compare the scavenging 
effect of SOD alone, mixtures of unconjugated SOD and catalase and 
SOD-catalase conjugates (prepared as in Example 1), on OH. radical levels 
stimulated by 0.1 mM FeCl.sub.3. Results are shown in Table 3. 
TABLE 3 
__________________________________________________________________________ 
Incubation Conditions Integrated OH. free radical values* 
__________________________________________________________________________ 
(%) 
1. x + xo + 0.1mM FeCl.sub.3 + SOD (42.4.mu.) 
100 
2. x + xo + 0.1mM FeCl.sub.3 + SOD (84.8.mu.) 
100 
3. x + xo + 0.1mM FeCl.sub.3 + SOD (127.mu.) 
100 
4. x + xo + 0.1mM FeCl.sub.3 + SOD (42.4.mu.) + CAT (6.mu.) 
80 
5. x + xo + 0.1mM FeCl.sub.3 + SOD-CAT (units as in 4) 
62 
6. x + xo + 0.1mM FeCl.sub.3 + SOD (84.8.mu.) + CAT (12.mu.) 
36 
7. x + xo + 0.1mM FeCl.sub.3 + SOD-CAT (units as in 6) 
22 
8. x + xo + 0.1mM FeCl.sub.3 + SOD (127.mu.) + CAT (18.mu.) 
26 
9. x + xo + 0.1mM FeCl.sub.3 + SOD-CAT (units as in 8) 
0-5** 
__________________________________________________________________________ 
*1 set as 100% 
**large error due to very low values 
x = xanthine and xo = xanthine oxide 
Samples 1, 2 and 3 showed that in the presence of iron and SOD, substantial 
amounts of OH free radical formed which were not abolished by increasing 
SOD levels. Catalase in the presence of SOD (Samples 4,6 and 8) decreased 
OH free radical formed, probably by decreasing H.sub.2 O.sub.2. 
In each case, it is seen that comparable levels of catalase conjugated to 
SOD (5,7 and 9) were more effective than free SOD and Catalase (4,6 and 8) 
presumably because H.sub.2 O.sub.2 formed by SOD was immediately converted 
to H.sub.2 O+O.sub.2 by the conjugated catalase thus inhibiting the 
formation of OH free radicals by the Haber Weiss reaction. 
SOD and catalase in conjugated form were more effective in scavenging free 
radicals than similar levels of the two enzymes in unconjugated form. 
Example 9 
A hanging working rat heart model of ischemia-reperfusion was set up as 
described by Lopaschuk et el., Circulation Research (1988), Vol. 63, p. 
1036. 
A 10 min Langendorff perfusion was initiated with Krebs-Henseliet buffer 
(pH 7.5) containing 11 mM glucose. Hearts were then switched to the 
working mode for 10 min with buffer containing 3% bovine serum albumin, 11 
mM glucose and 1.2 mM palmirate. Hearts were perfused at a left atrial 
filling pressure of 11.5 mm Hg and a hydrostatic afterload of 80mm Hg. 
When used, SOD and SOD-careless conjugate were added directly to the 
perfusate. Low dose SOD=100 U/ml; high dose SOD=1000 U/ml; for conjugates, 
same amounts of SOD were conjugated to a constant amount of catalase, the 
conjugate being prepared as in Example 1. 
Aerobic work continued under these conditions for an additional 10 min. The 
preload and afterload lines were then clamped inducing global ischemia. 
Following 30 min of global ischemia, the hearts were reperfused for 30 min 
then frozen with Wollenberger clamps cooled to the temperature of liquid 
nitrogen. 
Cardiac function was measured as described by Lopaschuk etal. 
The results are shown in Table 4 and FIGS. 4a to 4d. 
TABLE 4 
______________________________________ 
CARDIAC FUNCTION 
% Return to Base 
Experimental Group following ischemia 
______________________________________ 
Control 32 
Low Dose SOD 48 
High Dose SOD 20 (0-50)* 
Low Dose SOD-Catalase 
80 
High Dose SOD-Catalase 
80** 
______________________________________ 
Each value represents the mean from at least 6 hearts. 
*The range for the high SOD hearts was much larger than any of the others 
and of a total of 10 hearts there were 4 complete failures. 
**The value for the High SODCatalase is from only 4 hearts. 
As seen from Table 4, the lower dose of SOD gave significant protection 
compared to the control, but the higher SOD dose was very toxic to the 
heart preparation. When the lower dose of SOD was conjugated to catalase, 
the conjugate was much more protective of heart function than the 
equivalent amount of free SOD. A similar degree of protection was seen 
with the conjugate containing the high SOD dose, the additional toxicity 
seen with that level of SOD alone being completely eliminated. 
FIGS. 4a to 4c show the protective effect of low dose SOD-catalase 
conjugates compared with control perfusses. FIG. 4d shows the improved 
protective effect of low dose SOD-catalase conjugate compared with an 
equivalent low dose of SOD alone. 
SOD-catalase conjugates clearly permitted restoration of heart function to 
a much greater level after ischemia than SOD alone. The SOD-conjugate had 
a molecular weight of 450,000.+-.50,000 and was cleared in vivo at a rate 
50-100 times more slowly than the equivalent amount of free SOD. 
The present invention is not limited to the features of the preferred 
embodiments described herein, but includes all variations and 
modifications within the scope of the claims.