THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 263, No. 14, Issue of May 15, pp.

6884-6892, 1988
0 1988 by The American Society for Biochemistry and Molecular Biology, Inc Printed in U.S.A.

Superoxide Dismutase and CatalaseConjugated to Polyethylene Glycol

Increases Endothelial Enzyme Activityand Oxidant Resistance*
(Received for publication, September 3, 1987)

Joseph S. Beckman$, RobertL. Minor, Jr.5, Carl W. Whiten, John E. Repinell, Gerald M. Rosenil,
and BruceA. Freeman$
From the $Departments of Anesthesiology and Biochemistry, University of Alabama at Birmingham, University Station,
Birmingham, Alabama 35233, the §School of Medicine, University of Iowa, Iowa City, Iowa 52242, the ll Webb-Waring Lung
Institute, University of Colorado Health Sources Center, Denver, Colorado 80302, and the 11 Department of Pharmacology,
Duke University Medical Center, Durham, North Carolina 27710

Covalent conjugation of superoxide dismutase and drugs, radiation exposure, and ischemia (2, 3), has also stim-
catalase with polyethyleneglycol (PEG) increases the ulated the use of antioxidant enzymes as therapeutic agents.
circulatory half-lives of these enzymes from <lo min However, the experimental and therapeutic potentialsof su-
to 40 h, reduces immunogenicity, and decreases sensi- peroxide dismutase and catalase are limited by two factors.
tivity to proteolysis. Because PEG has surface active First, both proteins are rapidlycleared by kidneys (4,5 ) ,
properties and can induce cell fusion, wehypothesized leading to circulatory half-lives of only 6-10 min following
that PEG conjugation could enhance cell binding and intravenous injection. Second, reactive oxygen species diffuse
association of normally membrane-impermeable en- very short distancesbefore reacting with cellular components
zymes. Incubation of cultured porcine aortic endothe- (6), and neithersuperoxide dismutase nor catalase can pene-
lial cells with lZ6I-PEG-catalaseor ‘261-PEG-superox- trate across cell membranes. Thus, antioxidant enzymes can
idedismutaseproduceda linear,concentration-de-
pendentincreaseincellularenzymeactivityand not gain access to intracellular sitesof free radical generation
radioactivity. Fluorescently labeled PEG-superoxide to achieve effective pharmacological value.
dismutase incubated with endothelial cells showed a The renal clearance of superoxide dismutase and catalase
vesicular localization. Mechanical injury tocell mono- can be prevented by increasing theirmolecular weight through
layers, which is known to stimulate endocytosis, fur- covalent attachment of the inert linear polymer, monome-
ther increased the uptake of fluorescent PEG-super- thoxy-polyethylene glycol (PEG;’ Refs. 7-9). The structureof
oxide dismutase. Endothelial cell cultures incubated PEG isH-0-(CH,-CH,-0)”-CH, with n = 150 forPEG weigh-
with PEG-superoxide dismutase and PEG-catalase for ing 5000 Da. The free hydroxyl of PEG is conjugated to t-
24 h and then extensively washed were protected from amino groups of lysine with a bifunctional reagent such as
the damaging effects of reactive oxygen species de- cyanuric chloride. Typically, from 10 to15 of the 20 available
rived from exogenous xanthine oxidase as judged by amino groups on superoxide dismutase aremodified with PEG
two criteria: decreased release of intracellular W r - monomers of a n average molecular mass of 5000 Da each,
labeled proteins and free radical-induced changes in increasing the molecular mass of superoxide dismutase from
membrane fluidity, measured by electron paramag- 32 kDa to about100 kDa for the modified protein. Similarly,
netic resonance spectroscopyof endothelial membrane the molecular weight of catalase may be increased by 300%
proteins covalently labeledwith 4-maleimido-2,2,6,6- following PEG conjugation. Modificationby PEG blocks renal
tetramethylpiperidinooxyl.Addition of PEG andPEG- clearance and increases the circulating enzyme half-life from
conjugated enzymes perturbed the spin-label binding 6 min to 30-40 h in the rat(9). The inert natureof PEG also
environment, indicative of producing an increase in reduces the antigenicityof the native protein and inhibits the
plasma membrane fluidity. Thus, PEG conjugation to hydrolysis of protease-sensitive proteins such as catalase(8).
superoxide dismutase and catalase enhances cell asso- Additionally, the long term stability of enzymes in aqueous
ciation of these enzymes in a manner which increases
cellular enzyme activities and provides prolonged pro- solution is frequently increasedby PEG conjugation.
tection from partially reduced oxygen species. Polyethylene glycol is a surface-active molecule that is used
extensively to induce fusion for cell hybridization (10). Mem-
branes can absorb substantial quantities of PEG, binding 1
PEG molecule/l2 dipalmitoyl-phosphatidylcholine molecules
Controlled manipulation of cellular superoxide dismutase- in liposomes with a dissociation constant of 6 p M (11).Be-
and catalase-specific activities can help
define mechanisms of cause PEG constitutes up to two-thirds of the totalmolecular
tissue injury mediated by superoxide and hydrogen peroxide weight of PEG-conjugated enzymes, we have investigated the
(1).The toxicity of reactive oxygen species, whose rate of possible membrane association of PEG-conjugated enzymes
production can be amplified by pathological events including with cultured endothelialcells. Endothelial cells were used as
neutrophil activation, hyperoxia, metabolism of redox-active target cells in this study,because the vascular endothelium is
a significantsite of oxidant injury (12)and because the
* This work wassupported by National Institutes of Health Grants anatomical localization of the vascular endothelium provides
NS-23700 and NS-24275 (to B. A. F.), HL-28182 (to J. E. R.), and
HL-33550 (to G . M.R.) and by grants from the Health Effects
direct contact with circulating antioxidant enzymes. We re-
Institute (to B. A. F.) and the Alabama Affiliate of the American
Heart Association (to J. S. B.). The costs of publication of this article ’ The abbreviations used are: PEG, polyethylene glycol; FITC,
were defrayed in part by the payment of page charges. This article fluorescein isothiocyanate; HBSS, Hank’s balanced salt solution;
must therefore be hereby marked “advertisement” in accordance with Mal-6-TEMPO, 3-maleimido-2,2,6,6,-tetramethylpiperidinooxy~ W/
18 U.S.C. Section 1734 solelyto indicate this fact. S ratio, the weakly to strongly immobilized peak ratio.

6884

This is an Open Access article under the CC BY license.

 Enzymes
Antioxidant
Endothelial
Augmenting 6885
port that PEG conjugation of superoxide dismutase and cat- samples of cell homogenate by y-counting. Protein concentrations of
cell homogenates were measured by the method of Lowry et al. (22).
alase enhances cell association of these antioxidant enzymes, "Cr Release Studies-Endothelial cells were grown in 2.2-cm' well
alters membrane fluidity, and confers resistance to oxidant plates (Costar)and prelabeled with 3 pCi/ml 'lCr added to theculture
stress. medium for 4 h, washed 3 times, and then incubated for 24 h with
either native or PEG-catalase. The cells were then washed five times
EXPERIMENTALPROCEDURES and maintained in HBSS for the duration of xanthine oxidase expo-
sure. Xanthine oxidase (10 milliunits/ml) and xanthine (100 p M )
Materials-Pharmaceutical grade, bovine Cu,Zn superoxide dis- were added to HBSS, and the release of'lCr from cells into the
mutase was generously provided by Diagnostic Data Inc. (Mountain medium was measured 4 h later. The percentage of 51Cr release for
View, CA) and by Grunenthal GmbH (Aachen, Germany). The su- each treatment was calculated by subtracting the amount of"Cr
peroxide dismutase obtained from Diagnostic Data Inc. was dialyzed released by untreated cells (not exposed to xanthine oxidase) and
overnight against phosphate-buffered saline to remove sucrose added dividing by the additional amount of 'lCr released when untreated
as a preservative. Bovine catalase was obtained from Cooper- cells were exposed to xanthine plus xanthine oxidase.
Biomedical, Inc. Bovine milk xanthine oxidase was purchased from Spin-lubeling Measurements-Endothelial cell monolayers were
Behring Diagnostics. Tissue culturemedia were obtained from Gibco, first washed free of serum-containing medium with HBSS containing
and fetal calf serum was obtained from HyClone (Logan, UT). All 0.7 mM L-glutamine and 5 mM glucose, pH 7.4, and labeled for 3 h at
other reagents were obtained from Sigma. 37 "C in the same buffer with 100 p~ 4-maleimido-2,2,6,6-tetra-
Enzyme Assays-Superoxide dismutase was assayed by inhibition methylpiperidinooxyl (Mal-6-TEMPO) as reported previously (23).
of cytochrome c reduction, and units of activity are defined as given The labeled cells were washed free of unreacted labeling reagent,
by McCord and Fridovich (13). Catalase activity was determined by removed from flasks by scraping, and resuspended to a density of
the disappearance of 10 mM hydrogen peroxide in 50 mM K' phos- 400,000 cells/ml, which corresponds to about 0.4 mg of protein. The
phate, pH 7.0, monitored at 240 nm ( A c Z 4=~ 43.6 M" cm"; Ref. 14). labeling procedure did not affect cell viability or the rate of cell
Xanthine oxidase was assayed by monitoring the production of uric respiration. Immunoblot analysis using rabbit anti-Mal-6-TEMPO
acid at 295 nm (AcZs5 = 1.1 X lo' M" cm"; Ref. 15) at 25 "C. The IgG following sodium dodecyl sulfate-polyacrylamide gel electropho-
reaction contained 50 p~ xanthine, 100 p~ EDTA, and 50 mM K' resis of solubilized endothelial cell membranes showed predominant
phosphate, pH 7.8. Units of activity for catalase and xanthine oxidase labeling of two major membrane proteins (23). The EPRspectra were
are defined as 1 pmol product produced per min. obtained from a Varian Associates E9 spectrometer (Palo Alto, CA)
Preparation of PEG-Superoxide Dismutase and PEG-Catalase- operated at 9.5 GHz with 100-kHz modulation. Spectra from 0.5-ml
Cyanuric chloride (10 g) was dissolved in 100ml of chloroform, samples were recorded at room temperature in a flat quartz cuvette
filtered through Whatman No. 1 paper and evaporated to dryness with a microwave powerof 14 milliwatts and a modulation amplitude
immediately before use (16). Monomethoxy-polyethylene glycol (5000 of 0.63 gauss.
Da) was activated with cyanuric chloride and coupled to superoxide Statistical Analysis-The rate of PEG-conjugated enzyme uptake
dismutase and catalase by standard methods (7-9). The extent of by endothelial cells was determined by multiple regression. The rate
conjugation, measured by the number of reactive amino groups, was of change in the W/S ratio during exposure to xanthine oxidase was
determined by titration with trinitrobenzene sulfonic acid (17), and determined by linear regression with the difference between slopes
protein concentrations were measured by the Biuret method (Sigma from the regression lines determined by Tukey's post hoc procedure
technical procedure 540). Typically, about 60-70% of the available (24). Changes in the extent of 'lCr release from endothelial cells by
amino groups on superoxide dismutase and 60-80%of the amino catalase and PEG-catalase were determined by analysis of variance
groups on catalase were conjugated to PEG, and there was no loss of and Scheffe's post hoc test (25).
enzyme activity. Superoxide dismutase assay showed3300-3500
units/mg protein bothbefore and after PEG conjugation. Previously,
RESULTS
a 50% loss of superoxide dismutase activity was reported when 95%
of the available amino groups were modifiedwith PEG by the cyanuric Association of PEG-Catalase with Endothelium-Incubation
acid method (9). Using the same protocol, we found slightly less of cultured porcine aortic endothelial cells with lZ5I-labeled
modification (60-70%) without loss of superoxide dismutase activity
while retaining their long circulatory half-lives in rodents.' The PEG-catalase produced a linear, dose-dependent increase of
commercial preparation of PEG-superoxide dismutase, made with a cell-associated enzyme activity with time (Fig. 1).There was
succinylated PEG, is also only 50% modified and retains full activity.3 an initial rapid cell association of PEG-catalase within the
Fluorescent and Rudwuctive Labeling of Proteins-Proteins were first 4 h which was followed by a slower, continuous uptake
labeled with lZ5Ivia a solid phase oxidative reaction using 1,3,4,6- over the next 24 h. This pattern of PEG-catalase uptake was
tetrachloro-3a,6a-diphenylglycoluril (Pierce Chemical Co.; Ref. 18) described by the following equation:
and then exhaustively dialyzed against phosphate-buffered saline to
remove free lz5I.Fluorescence labeling of Cu,Zn superoxide dismutase Units = a,t[PEG-CAT] + a2[PEG-CAT] + a3 (1)
was accomplished by addition of 0.3 mg of fluorescein isothiocyanate
(FITC) to 1.0 ml of 10 mg/ml protein in 50 mM sodium carbonate, where units equal the catalase activity/mg cell protein meas-
pH 9.5, and stirred for 1 h at 4 "C while maintaining pH at 9.5 by ured at time t following the addition of PEG-catalase, [PEG-
addition of Na2C0, (19). Unbound FITC was removed by gelfiltration CAT] is the concentration of PEG-catalase added to the
chromatography on Sephadex G-25 eluted with phosphate-buffered
saline (150 mM NaCl, 10 mM K' phosphate, pH 7.4). medium, a, is the slow, continuous secondary rate of PEG-
Cell Culture-Porcine thoracic aortic endothelial cells were cul- catalase uptake/h, a2is the initial rapid phase of PEG-catalase
tured asdescribed previously (20). Third or fourth passage cells were uptake, and u3 is the endogenous catalase activity of control
grown to confluence in 25-cm2 flasks (Costar; Cambridge, MA) in cells. Multiple regression analysis of the datashown in Fig. 1
M199 medium with 10% fetal calf serum at 37 "C before use. Cells yielded a correlation coefficient of 0.93, and thevalues deter-
were passaged in a 1:3 split ratio with 0.05% trypsin and used by the
fourth passage to avoid changes in endogenous antioxidant enzyme
mined for a,, a2, and a3 are given in Table I. These values
activities (21). were used to calculate the fitted lines shown in Fig. 1 for each
Sample Preparation-Cells were washed three times with Hank's concentration of PEG-catalase. Thus, addition of 1 mg/ml
balanced salt solution (HBSS) before being removed from culture PEG-catalase to cell monolayers doubled endogenous catalase
flasks with a cell scraper (Costar), washed again in 5 ml of HBSS, activity within 4 h and resulted in an additional increase in
and sonicated in 1 ml of 50 mM K+ phosphate, 0.1% Triton X-100, activity of l l % / h over the next 20 h. This increase in both
pH 7.8. The sample was centrifuged at 15,000 X g for 30 min and the
supernatant assayed for either catalase or superoxide dismutase ac-
catalase activity and enzyme-associated radioactivity could
tivity. The amount of '"I-label uptake was determined from 150-pl not be removed after extensive washing of PEG-catalase-
treated cells.
* J. S. Beckman, R. L. Minor, Jr., C. W. White, J. E. Repine, and In the above experiment, the cell uptake of PEG-catalase
G. M. Rosen, unpublished data. measured by '1 radioactivity closely paralleled the increase
A. Abuchowski, personal communication. in catalase specific activity. The specific activity of PEG-
6886 Augmenting Endothelial Antioxidant Enzymes
12a
catalase was 20 times greater than could be accounted for by
the increase in catalase enzyme activity after 24 h of incuba-
tion (Table 11). The most likely explanation for this is that
1W
native catalase was avidlybound to anddegraded by endothe-
lial cells and that radiolabeled amino acids or peptides re-
mained associated with cell.
Association of PEG-Superoxide Dismutase with Endothe-
lium-Incubation of cells with 0.5 mg/ml PEG-superoxide
dismutase increased cellular superoxide dismutase-specific
activity by about 17%/h (Fig. 5). During the first 4 h, incu-
bation with 0.5 mgjml native bovine Cu,Zn superoxide dis-
mutase nearly doubled endothelial superoxide dismutase ac-
tivities from 4.4 f 0.8 to 8.2 f 0.8 units/mg cell protein.
Thereafter, the activity for native superoxide dismutase-
treated cells did not increase significantly, rising only to 9.0
20 f 0.6 units/mg cell protein after 24 h (Fig. 5 ) . As with native
catalase, Cu,Zn superoxide dismutase was predominantly in-
corporated as an inactive species, since there was a progressive
0 increase in the ratio of cpm of "'I/unit superoxide dismutase
in endothelial cells compared with medium counts/min of
lZ5I/unitsuperoxide dismutase (Fig. 6). If cells incubated with
INCUBATION TIME (HR)
native superoxide dismutase for 8 h were washed and placed
FIG. 1. Polyethylene glycol-catalase uptake by endothe- in fresh medium, the counts/min of lz5I/unit superoxide dis-
lium. The increase in catalase specific activity/mg cell protein is
shown for endothelial cell monolayers incubated with the following mutase ratio declined over the next 16 h to near control
concentrations of PEG-catalase: 0, 1.0 mg/ml; A, 0.5 mg/ml; 0, 0.1 values. In contrast, cells incubated with PEG-superoxide dis-
mg/ml; A, 0.05 mg/ml; and H, untreated cells. Regression lints were mutase showed a constant ratio of counts/min of '"I/unit
calculated from Equation 1 using the coefficients given in Table I for superoxide dismutase over endogenous superoxide dismutase
each concentration of PEG-catalase. Each point represents the mean activity that was the same as thespecific activity of "'I-PEG-
f S.E. of three replicates. superoxide dismutase added to the medium. Furthermore, if
medium containing PEG-superoxide dismutase was replaced
TABLEI with fresh medium after 8 h incubation, cellular superoxide
Analysis of the cell association of PEG-catalase by multiple regression dismutase activities remained elevated andthe ratio of
The data shown in Fig. 1 were fitted t o Equation 1 to determine counts/min of "'I/unit superoxide dismutase remained con-
the coefficients al, az,and a3.Each incubation time and PEG-catalase stant. When lysates of endothelial cells incubated for 24 h
concentration contained three replicates with a total of 39 observa-
tions used in the regression. The overall correlation coefficient was
with '2'I-labeled PEG-superoxide dismutase were chromato-
0.93, and all coefficients were significant at the 0.0001 level. graphed over Sephadex G-25, all of the radioactivity from the
Estimate
PEG-superoxide dismutase-treated cell lysates eluted in the
Coefficient
* S.E. Units void volume. These results show that PEG-superoxide dis-
2.5 _+ 0.24
mutase is resistant to degradation by endothelial cells to
a1 Units (mg PEG-catalase)"
h" (mg cell protein)" smaller peptides or amino acids.
a2 21 f 4.2 Units (mg PEG-catalase)" The uptake of FITC-PEG-superoxide dismutase could also
(mg cell protein)" be observed by fluorescence microscopy, demonstratinga
aa *22 1.5 Units
(me cell motein)" punctate fluorescence distribution pattern (Fig. 7). Mechani-
cal injury to the endothelium, produced by scratching the
catalase added to culture medium was 6.5 cpm of "'I/unit monolayer with the sealed tip of a glass Pasteurpipette,
catalase. When the radioactivity uptake by cells was plotted greatly stimulates pinocytosis in the endothelial layer that
against the increase in cell catalase activity (Fig. 2), it was grows into the"injured" area (26). Forty-eight h after injuring
found that 7.6 f 0.6 cpm of lZ51/unitcatalase was taken upby endothelium in the presence of FITC-PEG-superoxide dis-
the endothelial cells. From these data, we estimate that at mutase, the injured area showed numerous fluorescent
least 85% of PEG-catalase takenup by endothelial cells patches which, upon focusing through the cell, wereuniformly
retainedits activity, probably by electrostatic membrane- distributed at all depths. This was consistent with the uptake
protein interactions (29). of fluorescent protein into discrete subcellular vesicles which
Incubation of cell monolayers with 0.5 mg/ml native cata- might be endosomes and lysosomes.
lase increased catalase activity from 11units/mg cell protein Resistance to Oxidant Stress-The degree of protection
to 30 units/mg cell protein within the first 4 h (Fig. 3). Unlike afforded by superoxide dismutase or catalase against an oxi-
PEG-catalase-treated cells, longer incubation with native cat- dant stress imposed by xanthine plus xanthine oxidase de-
alase did not lead to further increases of cellular catalase pends upon the method chosen for assaying cellular injury.
activity. This difference was not due to the presence of PEG Wehave made use of two markers of cellular injury: the
alone, because the addition of 1%PEG tonative catalase did xanthine plus xanthine oxidase-induced release of 'lCr-la-
not increase endothelial catalase specific activities (Fig. 3). beled proteins from cells, and EPR detection of membrane
Thus, significant augmentation of cellular catalase activity organizational changes measured by changes in the peak
depended upon PEG being covalently bound to catalase. The amplitude of the two major binding environments represent-
uptake of radiolabel by cells incubated with '2'I-catalase was ing weakly and strongly immobilized proteins labeled with
far greater than thatobserved when cells were incubated with Mal-6-TEMPO. Our previous studies have shown that xan-
a similar isotope specific activity and protein concentration thine plus xanthine oxidase-induced 51Cr release from endo-
of '2'I-PEG-catalase (Fig. 4). Radiolabel incorporation of lZ5I- thelial cells is predominantly inhibitable by catalase (21),
 Augmenting EndothelialEnzymes
Antioxidant 6887

FIG. 2. Comparison of endothelial

cell-associatedradioactivityafter
incubation with ""I-PEG-catalase
compared to catalase specific activ-
ity for each culture flask for all of
the data shown in Fig. 1. The l i n e
represents the best fit by linear regres-
sion (rZ = 0.92).

0
0 0

I I I I

0 30 120 60 90

UNlTS CATALASVMG PROTEIN

18
t

16

14
e
12
X
Y

0 2 4

TIME (HR)
FIG. 3. Augmentation of endothelial cell catalase specific
21

activity. Cell incubations contained 0.5 mg/ml PEG-catalase (W),

native catalase (O),or native catalase plus 1%PEG (e) (average M.
5000). Control cell activities are shown (A).

while xanthine oxidase-induced changes in endothelial mem-

*t
0 E 0 2 4

INCUBATION TIME (HR)

24

brane protein mobility are inhibitable by superoxide dismu- FIG. 4. The radioactivity associated with endothelial cell
tase (23). monolayers incubated with 0.5 mg/ml medium of 'aaI-labeled
PEG-catalase (W) and lZsI-catalase(0).These values were meas-
The release of 51Crinto culturemedium from cells following ured in cell incubations reported in Fig. 3.
injury is analogous to the release of endogenous lactate de-
hydrogenase (27, 28). Control experiments showed that incu-
bation of PEG-superoxide dismutase or PEG-catalase(1 mg/ with concentrations of0.5 or 1.0 mg of PEG-catalase/ml
ml) with endothelial cells for 24 h(without exposure to medium for 24 h and washed free of enzyme-containing me-
xanthine plus xanthine oxidase) did not enhance 51Crrelease dium before xanthine oxidase exposure prevented 80% of the
from endothelial cells. Under the experimental conditions we 51Crrelease compared with untreated controls. Preincubation
employed, exposure to 10 milliunits of xanthine oxidase/ml with 0.5 or 1.0 mg of native catalase/ml medium reduced 'lCr
medium plus 0.1 mM xanthine induced lysis or detachmentof to a lesser extent. Addition of 1%PEG in the presence or
85% of the monolayer. Augmentation of endothelial cell cat- absence of native catalase (0.5 mg/ml) did not affect the
alase activity after 24 h preincubation with PEG-catalase amount of 51Crrelease compared with untreated cells.
increased resistance of endothelial cells to lysis induced by The protection provided by preincubation of cells with
xanthine oxidase (Fig. 8). Endothelial cell cultures incubated native catalase might bedue to the apparent electrostatic
6888 Augmenting EndothelialEnzymes
Antioxidant
TABLE I1
Percentages of enzyme added to the medium which was taken up by
endothelial cell monolayers after 24-hincubation
The catalase data were derived fromthe experiment shown in Figs.
3 and 4, while the superoxide dismutase data came from Fig. 6.
Percentage uptake
Enzyme Radio-
activity activity
PEG-catalase 0.07 0.12
Catalase 0.01 0.20
PEG-superoxide dismutase
0.10 0.13
Superoxide dismutase 0.01 0.04

I 1 I
0 8 16 24

INCUBATION TIME (HR)

FIG. 6. The ratio of cellular '''1 counts/min uersus units of
superoxide dismutase (SOD)activity taken up by endothelial
cells incubated with 0.5 mg/ml '261-PEG-superoxidedismu-
tase (W) or '"I-superoxide dismutase (a). After 8 h of incubation
with '251-PEG-superoxidedismutase or"'1-superoxide dismutase,
0 2 4 24 some flasks were washed three times and placed in fresh mediumto
observe the disappearance ofcell associated enzyme activities (dashed
TIME ( H R ) line).The "'I/unit superoxide dismutaseratios shown at time 0 reflect
the specific activities of the '251-labeled superoxide dismutase and
FIG. 5. The time course of increased cellular superoxide
PEG-superoxide dismutase addedto the media.
dismutase (SOD)specific activities after incubation with 0.5
mg/ml either native superoxide dismutase (a)or PEG-super-
oxide dismutase (B).Control cell activities are shown (A). teins. Part of the EPR spectrum of Mal-6-TEMPO was split
into two peaks depending upon the binding environments of
attachment of native catalase to membranes (29). Addition of Mal-6-TEMPO-protein conjugates. These binding environ-
native catalase to endothelial cells followed bywashing of the ments were characterized as being either weakly ( W ) or
cells increased the catalase specific activity from 11 & 1.2 to strongly ( S ) immobilized (Fig. 9A). The ratio of the peak
20 f 1.6 units/mg cell protein. We have found that addition heights (the W/S ratio), a measure of membrane disorder,
of catalase to cationic liposomes leads to membrane aggrega- increases as membrane fluidity becomes greater. Under the
tion (29). Modification of the surface change of catalase with reaction conditions employed here, the W/S ratio was 2.8 f
succinic anhydride greatly diminished the aggregation of li- 0.1 for all labeled control endothelial cells and stable for at
posomes, suggesting that electrostatic charges on catalase least 15 min in the EPR cuvette. Exposure of the cells to
mediate its association with membranes. either 1% PEG or 1 mg/ml PEG-superoxide dismutase in-
Preincubation of cells with PEG-superoxide dismutase did creased the W/S ratio from 2.8 to 4.0 with the W/S ratio
not protect endothelial cells from xanthine oxidase-induced remaining at 4.0 (in the absence of xanthine oxidase) even
51Crrelease, consistent with previous studies where that na- after extensive washing of cells treated with either 1% PEG
tive superoxide dismutase added to culture medium at the or PEG-superoxide dismutase. These results show a mem-
time of cell exposure to xanthine oxidase was not protective brane association and fluidity change induced by PEG alone
(28). Thus, formation of hydroxyl radical by the superoxide- or protein derivatives of PEG.
driven, iron-catalyzed Fenton reaction can be ruled out in the The oxidant stress imposed on Mal-6-TEMPO-labeled
present study, although trace amounts of iron were undoubt- endothelial cells by xanthine plus xanthine oxidase caused a
ably present in the HBSS. Ferric chloride added to the xan- steady increase in the W/S ratio of 0.83 f 0.02 min" (Fig.
thine oxidase and xanthine reaction in HBSS only slightly 9B). In all cases involving exposure to xanthine oxidase, the
increased 'lCr release (21), indicating either that sufficient sum of the peak heights for the weak and strong peaks were
iron is already present in the HBSS to mediate toxicity or unchanged during the experiment, indicating that the spin
that iron plays only a minor role. label was not being reduced or otherwise affected by the
Membrane Fluidity-Pretreatment of cells with PEG-su- experimental treatment. Analysis of supernatants of centri-
peroxide dismutase confers endothelial oxidant resistance fuged cellpreparations showed the increased mobility was not
using another index of injury based upon oxidant-induced due to the release of proteins from the cell surface into the
membrane fluidity changes. Membrane fluidity was measured medium. Previously, we have shown that 20% of the rate of
by conjugating a maleimide derivative of the spin label increase in the W/S ratio could be prevented by the addition
TEMPO to sulfhydryl groups of endothelial membrane pro- of superoxide dismutase (100 units/ml) during exposure to
 Augmenting EndothelialEnzymes
Antioxidant 6889
120

100

i
80
W
v)
a
w
-I
E 60
6
Lo

8
40

20

a
0 .5 1

[CATALASE]
FIG. 8. Increasedendothelial cell resistance to oxidant
stress followingtreatmentwithPEG-catalase. Endothelial
monolayers were prelabeled with %r and then incubated for 24 h
with 0.5 mg/ml either native (0) or PEG-catalase (M). The cell
monolayers were then washed five times and placed in HBSS. Xan-
thine oxidase (10 milliunits/ml) and xanthine (100 PM) were added
to themedium and therelease of 51Crby cells was measured 4 hlater.
Each point represents the mean f S.E. of eight replicates. At concen-
trations a t and above 0.4 mg/ml, the differences in 51Crrelease
between PEG-catalase- and catalase-treated cells were significantly
different at the0.05 level.

dition of superoxide dismutase to suspensions of PEG-super-

oxide dismutase-pretreated cells during xanthine oxidase ex-
posure did not enhance protection against membrane fluidity
changes provided by PEG-superoxidedismutase pretreatment
alone (Fig. 10). Although the initialW/S ratio of labeled cells
was increased by 1%PEG, preincubation with 1%PEG did
not affect the rate of change in the W/S ratio caused by
xanthine oxidase-derived free radicals or the protection af-
forded by native superoxide dismutase present during the
xanthine oxidase exposure. Because catalase previously did
not attenuate xanthine oxidase-induced membrane fluidity
changes when added either alone or with superoxide dismu-
FIG. 7. Cell uptake of FITC-labeled PEG-superoxide dis- tase (23), no EPR experiments were performed with PEG-
mutase. Endothelial monolayers were incubated with 1 mg/ml FITC-
PEG-superoxide dismutase in MI99 medium plus 10% fetal calf
catalase.
serum for 48h. Immediately before adding FITC-PEG-superoxide
DISCUSSION
dismutase, the monolayer was scored with the sealed end of a sterile
Pasteur pipette (upper panel).This removed a narrow strip of cells We have shown that conjugation of superoxide dismutase
which was completely recolonized 48 h later (middle panel). The and catalase to PEG enhanced uptake of the active form of
middle panel was photographed from a different location than the
upper panel but is superimposable with the lower panel. After the 48-
these enzymes by cultured endothelial cells and that prior
h incubation, the Petri plates were washed with HBSS three times. incubation of cells with these enzyme conjugates provided
The lower panel shows the punctate distribution of fluorescence greater resistance to oxidant stress than that afforded by
within the endothelial cells. The region with intense fluorescence native enzymes. Three possibilities could account for the
dots in the lower panel corresponds to part of the area scraped 48 h augmentation of endothelial cell enzyme activity following
before. incubation with PEG-conjugatedenzymes: (a) direct penetra-
tion of membranes by PEG-enzymes, ( b ) binding of PEG-
xanthine oxidase (23). In the present study, we found that enzymes to membrane surfaces, and (c) uptake of PEG-
pretreating endothelial cells with 1 mg/ml PEG-superoxide enzymes by endocytosis (Fig. 11).Polyethylene glycol conju-
dismutase for 24 h, followed by washing, was as protectiveas gation of proteins probably does not mediate migration di-
that afforded by a much greater concentrationof superoxide rectly through cell membranes. Horseradish peroxidase be-
dismutase present during exposure to xanthine oxidase. Ad- comes solublein benzene when conjugatedto PEG,suggesting
 6890 Enzymes
Antioxidant
Endothelial
Augmenting

15
r t 1% PEG + PEG-SOD
FIG. 10. The effect ofpretreatment of endothelial cells with
1 mg/ml PEG-superoxide dismutase (SOD) or with 1% PEG
on the rate of change in the W/S ratio following exposure to
12.5 xanthine oxidase plus xanthine. Cell exposures also had native
superoxide dismutase (300 units/ml) added directly to the cuvette
(open bars) to establish the maximum protection that could be pro-
vided by superoxide dismutase. Reaction conditions for exposure to
xanthine oxidase are described in Fig. 9. The rates of change in the
10
W/S ratio were determined by linear regression, and the standard
errors of the slopes are shown.
0
c-
a 7.5
The binding of PEG-enzymes to cell membranes does not
U explain the continuous uptake of enzyme conjugates by endo-
cn thelial cells over 24 h because the binding of PEG to mem-
5 branes shouldbe rapidly saturated (11).
5 The third explanation entails the uptake of PEG-conju-
gated enzymes by endocytosis from themedium. In confluent
stationary phase bovine aortic endothelial cells, the rate of
medium uptake by endocytosis is reported tobe 50 nl h"/106
2.5 cells (26). If we assume that this rateof endocytosis is similar
in cultured porcine endothelium and take into account cell
number and medium volume, between 0.06 and 0.09% of the
medium would be taken up by cells by endocytosis in 24 h.
0 1 1 I 1 I I The fraction of cellular uptake of both PEG-superoxide dis-
0 2 4 6 8 10 12 mutaseandPEG-catalasepresentinculture medium was
0.07-0.13% in 24 h (Table 11), consistent with the calculated
TIME (MIN) rate of uptake by endocytosis. The punctate distribution of
FIG. 9. A, the EPR spectrum of Mal-6-TEMPO-labeled endothe- fluorescence observed inendothelial cells incubated with
lial cells is shown. B, the time-dependent changes in the W/S ratio FITC-labeled PEG-superoxide dismutasealso suggests that a
are shown for control cells (O),cells exposed to xanthine oxidase (10
milliunits/ml) plus 100 pM xanthine (+), cells pretreated with 1% vesicular uptake process plays a major role in the uptake of
PEG and exposed to xanthine oxidase plus xanthine (A),and cells PEG-conjugated proteins. Mechanical injury to the mono-
incubated in the presence of 300 units/ml superoxide dismutase and layer, known to stimulate endocytosis (26), considerably aug-
xanthine plus xanthine oxidase (m). The lines were fit by linear mented the uptakeof FITC-PEG-superoxide dismutase, sup-
regression. porting endocytosis as a contributing factor in uptake of PEG-
conjugated enzymes.
that enough hydrophilicresidues aremasked by PEG to The difference in the rate of native and PEG-conjugated
permitan otherwisehydrophilic proteintopartitioninto enzyme uptake by endocytosis can be explained by the in-
hydrophobic solvents (30). While PEG itself is quite soluble creased resistance of PEG-conjugated enzymes to proteolysis.
in aromatic solvents such as benzene, it precipitates rapidly Because PEG-protein conjugates are resistant to proteases
in alkane solvents, a fact made use of in the synthesis of (7), they can survive within lysosomes, yielding higher cell-
activatedPEG forcoupling toproteins(7).Hence,PEG- associated specific activities. Native catalase added to either
derivatized enzymes would not be expected to be soluble crude endothelial cell lysates or delivered to endothelial cells
within the aliphatic core of phospholipid membranes and via liposomes is degraded with a half-life of 2-3 h (31). On
therefore areunlikely to traverse the membranecore. the other hand, superoxide dismutase is more resistant to
Polyethylene glycol avidly associates with membranes in proteolysis and liposome-delivered superoxide dismutase was
the phospholipid headgroup region ( l l ) , which might allow far more stable in endothelial cells than catalase (20). How-
anchoring of PEG-conjugated proteins to exofacial plasma ever, Cu,Zn superoxide dismutase loses its zinc cofactor when
membrane surfaces. Evidence in favor of this concept is the exposed to an acidic pH and becomes susceptible to protease
increased W/S ratio of Mal-6-TEMPO-treated endothelial degradation (32). Thus, native superoxide dismutase present
membranessecondarytoincubationwithPEG-superoxide in acidic endosomal and lysosomal compartments should be
dismutase, suggestingsomeinfluence is exertedupon cell degraded. Inthepresentstudy, degradation of both cell-
membranes by superoxide dismutase after PEG conjugation. associated native superoxide dismutase and catalasewas ob-
 Augmenting EndothelialEnzymes
Antioxidant 6891

FIG. 11. Three possible mecha-

nisms for PEG-conjugated antioxi-
dant enzyme uptake by endothelial
cells. Direct membrane penetration of
PEG-enzymes due tothe amphiphilic
nature of PEG is considered to be un-
likely because PEG would be insoluble
in the alphatic core of the membrane.
However, evidence suggests that PEG-
conjugated proteins may bind to thepo-
lar head groups of phospholipid mem-
branes as well as being taken up by en-
docytosis. The same mechanisms apply
equally to PEG-catalase. SOD, superox-
ide dismutase.

served, as evidenced by the continuous increase in the ratio tation of cellular antioxidant activities. Furthermore, areasof
of counts/min of Iz5I/unitenzyme in cells incubated with lZ5I- reversibly injured endothelium in vivo may concentrate even
labeled proteins (Figs. 4 and 6). Thisratio for PEG-superoxide greater levels of PEG-conjugated enzymes due to injury-
dismutase- and catalase-treated cells remained almost the enhanced endocytic processes (26).
same as thespecific activity of the PEG-superoxide dismutase Our results suggest that anadditional consequence of PEG-
and PEG-catalase added to the medium, even after 24 h of conjugation to proteins is the enhancement of protein asso-
incubation with the endothelial cells. ciation with cells. Uptake of PEG-conjugated enzymes by
On balance, there is evidence to support cell uptake of other cell types almost certainly will occur in uiuo as well.
PEG-enzymes by both membrane binding and endocytosis. The prolonged treatment with PEG-conjugated adenosine
These two explanations are not mutually exclusive and could deaminase in children afflicted with severe combined immu-
even complement each other. For example, the binding of nodeficiency disease due to adenosine deaminase deficiency
PEG-enzymes to cell surfaces may enhance endocytosis, led to restoration of immune function after 6 months (37).
thereby increasing rates of endocytic uptake. Further studies Conceivably, uptake of PEG-adenosine deaminase by imma-
are required to differentiate between PEG-enzyme uptake by ture lymphocytes may have contributed to the intracellular
endocytic bulk phase transport of protein conjugates in solu- removal of inhibitory metabolites in addition to those secreted
tion, or the internalization of PEG-enzymes bound to plasma into plasma, enabling these cells to differentiate and restore
membrane, whichwould then be distributedin endocytic immune function.
vacuoles and throughout the cell by membrane flow (33).
Acknowledgments-Both Diagnostic Data Inc. (Mountain View,
Can PEG-conjugated antioxidant enzymes entrapped CA) and Grunenthal GmbH (Aachen, Germany) generously provided
within endosomes or lysosomes also provide protection Cu,Zn superoxide dismutase for these studies. We thank Zermeena
against intracellularly generated superoxide and hydrogen Mirza and M. Kathy Cunningham for their expert assistance in the
peroxide, since these enzymes are separated from the cyto- culture of endothelial cells.
plasm by a membrane barrier? Endosomes and lysosomes
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