Reaction of autoxidation products of penicillamine with myeloperoxidase

BIOCHEMICAL

Vol. 169, No. 3, 1990

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1158-l 163

June 29, 1990

REACTION

OF AUTOXIDATION PRODUCTS WITH MYELOPEROXIDASE Leah A. Marquez

OF PENICILLAMINE

and H. Brian Dunford

Department of Chemistry, University of Alberta Edmonton, Alberta, Canada T6G 2G2 Received

May 18,

1990

SUMMARY: Spectral evidence is presented which shows that penicillamine is able to initiate the formation of the oxidized intermediates of myeloperoxidase in the absence of exogenous hydrogen peroxide. The autoxidation of penicillamine presumably produces superoxide which dismutates spontaneously to form hydrogen peroxide. Thus, the formation of both compounds II and Ill of myeloperoxidase was observed. We also report that penicillamine can directly reduce cytochrome c and therefore, it could possibly act as a one-electron donor to myeloperoxidase. 01990

Academic

Press,

Inc.

Penicillamine rheumatoid

(8,8-dimethylcysteine)

used

arthritis and a wide range of other human diseases

still remains

obscure.

Several

(2-4). More

recently,

studies

neutrophit

investigations were

myeloperoxidase

penicillamine interaction

on the

system

chlorinating

have focused

performed

(5-6).While

could

autoxidize

these

oxidation

reactions

adverse

effects (7) which

We report

penicillamine

was not studied.

here

Moreover,

hydrogen

the first evidence

its mode of action

investigated

in the presence For example,

peroxide

the use of penicillamine

for the interaction

of

function

of the drug with the human

studies

could be due to some reactions

with myeloperoxidase

treatment

on its effect on leucocyte

ability of myeloperoxidase

to form enough

for the

(1). However,

on the interaction

of the enzyme with the drug itself was not explored.

penicillamine

drug.

is extensively

mediated

the effect

of H202,

of

the

the possibility

that

to drive its own enzymatic has been linked to some by free radicals

of the autoxidation

from the

products

of

from bovine spleen.

MATERIALS AND METHODS Bovine spleen myeloperoxidase was prepared as described previously (8-10). The RZ ) of the samples used in this study was 0.82 or greater. The concentration nm’A 280 nm

tA430 of the enzyme was calculated using a molar absorptivity of 178 mM-t cm-l at 430 nm (I 1). Crystalline superoxide dismutase and catalase were obtained from Sigma Chemical Co St Louis, .-I at USA). The concentrations were calculated using molar absorptivities of 15.9 mM 4 cm 265 nm for superoxide dismutase (12) and 3.24 x 105 M“ cm-’ at 405 nm for catalase (13). Cytochrome c (horse heart) and D-penicillamine were also purchased from Sigma. Aqueous solutions of D-penicillamine were always freshly prepared. Hydrogen peroxide (30%) was obtained from BDH Chemicals and the concentration of diluted solutions were determined spectrophotometrically using a molar absorptivity of 39.4 M-l cm“ at 240 nm (14). 0006-291X/90 Copyrighr All rights

$1.50

0 1990 by Academic Press, Inc. of reproduction in any form reserved.

1158

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BIOCHEMICAL

169, No. 3, 1990

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Spectrophotometric measurements were made on a Cary 219 spectrophotometer equipped with thermostatted cuvette holders. All experiments were performed at 25.0 + 0.5 OC and ionic strength of 0.1 M due to the contribution of phosphate buffer, pH 7.2. The anaerobic experiments were performed in cuvettes sealed with rubber caps. Solutions were made anaerobic by flushing for 20 minutes with nitrogen (Linde, Union Carbide) purified by passage through an oxygen trap (Oxiclear, Dow Chemical Co.). RESULTS Figure 1 depicts what happens D-penicillamine

without

the enzyme compound

exogenous

was converted II or compound

hydrogen

penicillamine, absorbance

the Soret maximum

of catalytic

sharper

(Fig.

28).

peak

myeloperoxidase-penicillamine incompletely

which

of superoxide

of myeloperoxidase

both

to distinguish

as

back to the native state

of compound

and a large excess of

nm was shifted

II (Fig. 2A). This intermediate dismutase

catalytic amounts

of native myeloperoxidase

to its oxidized

to 455

nm, the

was found to be of catalase

were

intermediate

as

430 nm and the formation

of a shoulder

at about

that the peak at 625 nm (characteristic

of compound

III) is

superoxide

were

system,

dismutase

compound

(Fig. 2C). All of the above experiments

and

II formation

catalase

still occurred

were performed

added

to the

albeit slowly

in the presence

Reaction of penicillamine with bovine spleen myeloperoxidase. Spectrum A was obtained by putting 0.75 pM myeloperoxidase in 0.1 M phosphate buffer at pH 7.2. Spectrum B was obtained after adding 25 eq of penicillamine to the reaction mixture in A. Each scan period is 3 min 30 s. 1159

and

of oxygen.

Wavelength (nm) Fig. 1.

of

period of 3 min 30 s

is hard

dismutase

at 430

If instead of superoxide

it is noticeable When

intermediate(s)

amounts

by the peak that still persists at around

450 nm. However,

Within a scanning

upon the addition

points (spectra not shown).

added, there is only partial conversion shown

myeloperoxidase

III. The decay spectra of the intermediate(s)

characteristic

stable for several minutes.

peroxide.

to (an) oxidized

did not show a single set of isosbestic In the presence

to native spleen

BIOCHEMICAL

Vol. 169, No. 3, 1990

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

0.16

A

1

g

0.10

C

x 5

r”

2

c

0.04

L

+

I 400

I 500

Wavelength

I 600

i

I

I

" 400

500

I

600

Wavelength

(nm)

(nm)

C

i; i ‘00

Wavelength

(nm)

Fig. 2. Effects of superoxide dismutase and catalase on the reaction of penicillamine with myeloperoxidase. A) To 0.75 pM myeloperoxidase in 0.1 M phosphate buffer at pH 7.2 was added 70 nM superoxide dismutase. B) Same conditions as A) except that 20 nM catalase was added instead of superoxide dismutase. C) Both superoxide dismutase (70 nM) and catalase (20 nM) were added to 0.75 pM myeloperoxfdase in 0.1 M phosphate buffer at pH 7.2. The succeeding spectral scans for all cases were taken 3 min 30 s of each other after addition of 75 uM penicillamine. The arrows indicate the direction of absorbance changes with increasing time.

When reaction

the addition was observed

of penicillamine (spectra

to myeloperoxidase

was carried

out anaerobicallly

no

not shown).

The effect of penicillamine native enzyme was also investigated

on compound

II formed

by adding

under both aerobic and anaerobic 1160

hydrogen

conditions.

peroxide

In the

to

Vol.

169,

No.

3, 1990

BIOCHEMICAL

AND

I 600

700

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

0.06 8 cs 1 0.04 Q 0.02

0

3

I 400

I 500

Wavelength

4

I

,

I

400

500

600

Wavelength

(nm)

(nm)

Fig. 3. Effect of penicillamine on the rate of decay of myeloperoxidase compound II to native enzyme. To 0.5 uM myeloperoxidase was added 100 u-M penicillamine. Compound II was formed by adding 10 KM’ H202 to the reaction mixture under A) aerobic and B) anaerobic conditions. The arrows indicate the direction of absorbance changes with time.

I ’ Al 500 Wavelength

I

I

550

600

(nm)

Fig. 4. Reduction of cytochrome c by penicillamine. The initial reaction mixture contained 5 PM ferric cytochrome c to which 250 pM penicillamine was added. The same spectral changes were observed under aerobic conditions, anaerobic conditions and aerobic conditions in the presence of 0.10 uhf superoxide dismutase. The spectral scans were taken 4 min 30 s after each other; the arrows indicate direction of absorbance changes with increasing time. 1161

700

Vol.

BIOCHEMICAL

169, No. 3, 1990

presence

of oxygen,

compound

II undergoes

3A). On the other hand, when oxygen faster

RESEARCH COMMUNICATIONS

normal spontaneous

is absent, the reversion

decay to the native state (Fig. of compound

II to native form is

(Fig. 38). To explore

further

form of cytochrome reduced

dismutase

ability of penicillamine,

Upon addition

by the increase

c was observed

superoxide

the reducing

c was studied.

as shown

cytochrome (Fig.

AND BIOPHYSICAL

of penicillamine

in the intensity

under both aerobic

its reaction to cytochrome

of the peak

and anaerobic

was added to the system, cytochrome

with the oxidized c, the latter was

at 550

conditions.

nm. Reduction

Moreover,

c was still reduced

of

even when

by penicillamine

4). DlscussloN The

oxidizing

results

equivalents

of exogenous

presented

peroxide,

(Fig. 1). The concentration

radical

II in the presence mixture

and the hydrogen

peroxide

able

intermediate(s)

found in the blood samples autoxidizes

formed

to form a thiyl

to form compound can then oxidize

The spectra of the oxidized

II and III (Fig. 1). The presence

species thus appear

of more than one intermediate

was added,

II was obtained

stable (Fig. ZA), presumably instead of superoxide

because

dismutase,

dismutation

superoxide

dismutase

and catalase

2C). It can be inferred rapidly dismutated acted

upon by catalase

02-’

compound

is faster and thus more H202

dismutase II was more is formed.

formed

inhibit the formation from the autoxidation

oxidizes

penicillamine

If

Ill is formed as indicated by

which reacts with the enzyme to form compound

to form O2 which

is also

of a peak at 455 nm (Fig. 28). The presence

did not completely

that whatever

to H202

and the resultant

catalase was added, more compound

the sharper peak at 625 nm and the absence

the native

to be that of a

point in the decay spectra. When superoxide

yield of compound

Ill or it

I is also formed but rapidly decays to compound

verified by the lack of a single isosbestic a greater

to provide

In the absence

to its oxidized

with the drug (15). Penicillamine

of excess H202,

of compounds

is converted

used was that normally

II. Most probably compound

iS

by myeloperoxidase.

can react directly with myeloperoxidase

spontaneously

enzyme to compound

that penicillamine

catalyzed

myeloperoxidase

treated 02“

indicate

reactions

of penicillamine

patients

and superoxide.

can dismutate

clearly

to initiate oxidation

hydrogen

from rheumatoid

here

of compound

of both II (Fig.

of penicillamine

is

II. Excess H202 is

and the cycle of reactions

continues. AS

if penicillamine

expected

is added after compound

but in the absence absence

from the above mechanism,

acts

as a one-electron

and the thiyl radical while in the presence state concentration

II formation,

of 02 the decay of compound

of 02 penicillamine

H202

necessary

in the absence of 02, no reaction takes place. compound

if 0,

II to native state is hastened. donor to compound

of 02. autoxidation

lt.

is present

Possibly,

in the

II to form native enzyme

of penicillamine

for the formation of compound 1162

lt is stabilized

leads to a steady

Vol.

169, No. 3, 1990

BIOCHEMICAL

The fact that penicillamine ability to reduce

ferric cytochrome

agent in the system.

is capable

of superoxide

reduced

by penicillamine.

of acting as a one-electron

c. It is therefore

Even in the absence

presence

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

dismutase

not only 02-m capable

of 02 (where

( which

dismutates

second,

first, penicillamine penicillamine

may directly

then reacts with molecular We do not have direct

myeloperoxidase, the chlorination

due

any 02-m formed)

radical

releases

02-’

Acknowledoment: and Engineering

myeloperoxidase

Thus, no reaction

for the formation

to the interaction

of the autoxidation

was suggested

(16) which

adverse

leads to further

reactions.

hydrogen

This work was supported Research Council of Canada.

by operating

enzyme

which

conditions.

of ferro-myeloperoxidase,

however

c. products

of penicillamine

explanation

and the reducing Similarly,

peroxide

two possible

under anaerobic

as a possible acceptor

c is still

with the 02-m formed;

II and Ill form. While these two intermediates

II can act as a one-electron of mediating

reacts

was observed

agent for cytochrome

(which

cytochrome

to form the ferrous

is a reducing

reaction capable

reduce

to form) or in the

III could form through

then myeloperoxidase

evidence

both compounds

effect (6)) compound free

oxygen.

spectral

we show that penicillamine Thus,

autoxidizes

of acting as reducing

no 02-m is expected

It is also evident from these results that compound pathways:

donor is shown by its

are inactive

7. 8. 9. IO. 11. 12. 13. 14. 15. 16.

the decay

becomes

of compound

a III

production.

grant A1248

from the Natural Sciences

Feltkamp, T.E.W. (1979). Stand. J. Rheum. (suppl) 28. Chwalinska-Sadowska, H., and Baum, J. (1976) J. Clin Invest. 58, 871-879. Binderup, L., and Arrigoni-Martelli, E. (1979) Biochem. Pharmacol. 28, 189-192. Maini, R. N., and Berry, H. (1981) Clin. Pharmacol.Ther. Ser. 1, l-310. Matheson, N. R. (1982) Biochem. Biophys. Res. Comm. 108, 259-265. Cuperus, R. A., Muijsers, A. O., and Wever, R. (1983) Biochim. Biophys. Acta 749, 18-23. Stein, H. B., Patterson, A. C., Offer, R. C., Atkins, C. J., Teufel, A., and Robinson, H. S. (1980) Ann. Intern. Med. 92, 24-29. Davis, J. C., and Averill, B. A. (1981) J. Biol. Chem. 256, 5992-5996. Davis, J. C., and Averill, B. A. (1984) Inorg. Chim. Acta 93, L49-L51. Ikeda-Saito, M. (1985) J. Biol. Chem. 260, 11688-11696. Agner, K. (1958) Acta Chem. Stand. 12, 89-94. Briggs, R. G., and Fee, J. D. (1978) Biochim. Biophys. Acta 537, 86-99. Samejima, T., and Yang, J. T. (1963) J. Biol. Chem. 238, 3256-3261. Nelson, D. P., and Kiesow, L. A. (1972) Anal. Biochem. 49, 474-478. Saetre, R. and Rabenstein, D. L. (1978) Anal. Chem. 50, 276-280. Metodiewa, D., and Dunford, H. B. (1989) Arch. Biochem. Biophys. 272, 245-253.

1163

in

for its therapeutic substrate

REFERENCES 1. 2. 3. 4. 5. 6.

with