140 B*,~'h:mt~a et Bmphv~wa ,4cta. 1037 ~ 1990) 140-146 P l,~vze ~

BBAI+RO 3354'9

The chlorinating activity of human myeloperoxidase, high initial activity at neutral pH value and activation by electron donors

K . W . M . Z u u r b i e r , A .R.J . Bakken i s t , R. W e v e r a n d A.O. Mui j se r s

Lat~ratorv o/B~othenu~lO" and B,olevhncdogwal Cenlrt', Unlt'¢rs~tr of Amsterdam. Amaterdam t The" Netherland~

(Received lq September 1989,)

Ke~ ~.ard,,: M',eloperoxid0_,,c: Compound I I ; Chlorinating acti',ity: A~orb~c acid: 5-Armnosalic,dic avid

The steady-state activity of myeloperoxidase in the chlorination of ~ i m e d o n e at neutral pH ~as investigated. Using a Mopped-flow spectrophotometer we were able to ~ that the enzymic activity at pit 7.2 rapidly declined in time. Dm-ing the first 50-100 ms after addition of H 2 0 2 to the enzyme, a turnover number ol abma 3211 s - t per haem was observed. However, ff;~ activity decreased rapidly to a value ol abmtt 25 s - i after I s. This ~ that in daxf~nd stea~-state activit~ measurements, the real activity ol the enzyme at neutral pH is grossl) tmdefestimated. By |ollowiz~ the transient spectra of myeloperoxidase during turnover it was shown that the decrease in activity, was lwol~ by the formation of an enzymically inactive tocm of the e n ~ m e , Comimund I!. As demme;lrated befoce (Bolsdzer. B.G.J,M., Zoutberg, G.R., Cuperus, R.A. and Wever, R. (1964) Biochim. B i o l ~ x . Acta 784, 189-191) ~ snob as a.~c(~bie acid and ferro~~'dde convert C o m i m ~ IL which i~'tmwlates during turnover, into at'live , w y ~ x i d - a ~ . Activity measurements in the Wesetw¢ ol ascocbi¢ acid showed, indeed, that the ntoderale eazymi¢ aclivi~j was higher t'han in the absence ol ascocbic acid. With ~minosalicylic acid present, however, the myetopemxidase activity remained at a much higlwr level, namely aboul 15G s - ~ ~:: b_~_~ during the ~ interval |ram 100 ms to 5 s after mixing. From combined stopped-flow/rapid-scan experiments during turnover it ~ t e clear that in the pcesenee e l 5-aminosalicylic acid the initially formed Compotmd il was rai~lly convertt~ INmtk to uative en,~yme. Prestemlb,-state experiments :~owed that S-aminosalicylic acid reacted with Compound ii with a K z of 3.2 • l0 s M - t . s - I whereas lot, a.,,,corbic acid a K: of 1.5.104 M - t . s - t was measllred at pH 7.2. In the pecsence of S-aminosalicylic acid during the time interval in which the m)eloperoxidase activity remained consta:tt, a K~ to¢ H z O z at pH 7.2 was determined of about 30 p M at 200 mM chloride. In the absence of reduc(ants the same value was found dining the first 100 ms after addition of H 20 z to the enzyme. The physiological consequences o! these fi.-tdings are discussed.

Introduction

Ncutrophils are invol~ed in the defence mechaldsms o! the bt~ly against invading micro-organisms, since the} phag~.'ytose and kill microbes. Myeloperoxidase {donor : hydrogen-peroxide o~adoreductase, EC 1.I 1.1.7) ~s one of the granular enzymes of the polymorpho- nuclear leukocyte [1-4 I. After phagocytosis, myeloper- oxidase is excreted into the phagosome [2]. From chlor- ide. which is always present, and H_,O:, which is gener- ated from O,- pr.,-,duced by NADPH oxida_~e [2,5-.9], mycloperoxidase is able to prtaluce the bactericidal agent hypochlorous acid 110-14],

C,me',pondence A.C,. Mmjsers. Lal'~.rato~ o[ Bi,~:henuxt~. L m,.cr- ,It', ,ff Am,,terdam. Meil-rtgdrccf 15. 1105 AZ Amsterdam. The Ncthcrlaadx.

In the ~atalytic reaction cycle, native myeloperox- idave reacts with H202 to form Compound 1 [15-17]. Compound I oxidises chloride to hypochlorous acid

ith concomitant regeneration of native enzyme [14.15,18-20]. At neutral pH, ho,vever, Compound i may react with a second molecule of H:O2, or with other suitable electron donors, if present, to yield Com- pound Ii [21-22], which is an inactive form of myeloperoxidase with regard to formation of hypochlo- rous acid [15,23]. Because of this progressive inactiva- tion of myeioperoxidase at neu:ral pH. investigation of the kinetic parameters of rr/eioperoxidase at this pH was almost impossible. Another intrinsic complication in measuring the chlorinating actbity of myeloperoxid- ase at neutral pH is the fact that a scavenger of and indicator for h)pochlorous acid formation is a reducing agent and thus has potential to interfere ~ith the cata- lytic reaction b~ reducing Comv~,und I to Compound

(,1C74~3' ~).'$U3.SLI ' I~h) Eb, e~,er ."~.mnce Publishers B ~ (~honledllca! L~,,1,qon)

ll. For the monochlorodimePone u.,~d in the pr~,,~nt study, this has recently b e e n shown by Keltic and Winterbgurn 124]. As shown before, me reductant ascorbk, acid is able to convert Compound I1, which accumulates during turnover, to native enzyme [23,25]. However. at neutral pH. with the use of classical meas- urement methods, the activity of myeioperoxidase in the presence of ascorbic acid decreased in time and accu- rate _ae.lermination of kinetic parameters, such as maxi- mal velocity and Km for the two substrates H2Oz and chloride, wa~ no: po--~;.ble.

Recently it has been derr~',nstraled that during the first 2 rain after formation of the phagocytic vacuole. the pH within this vacuole rises to a value of 7.75, and that afterwards acidification takes place [26]. Therefore evaluation of the kinetic parameters at neutral pH is of importance. In this study the course of the turnover rate and compound formation of myeloperoxidase at pH 7.2 was investigated in the ab~nce and presence ol ascorbic acid. usir.g a stopl:~d-flow/rapid-scan ~pectrophotome- ter. Since it was discovered thal 5-a, ,nosali ',he acid had a m u c h greater stimulating effect on the rate of mycioperoxidase than ascorbic acid, the myeloperoxi- dase kinetit,~ were a l~ studied in the pre~nce of 5- aminosalicylic acid. Under well-defined conditions it was possible to determine a K,, for H_.O:.

Maler ia i s and M e t h o ~

Myeloperoxidase was purified from h u m a n leuko- cytes, following the method d e ~ - r i b e d in Ref. 27. Myeloperoxidase preparations with a ,;28 nm/280 nm absorption ratio higher than 0.75 were u,,~cl for the experimep'g. The concentration of myeloperoxida_,,e was deternuned w i t h an absorption coefficient of 89 m M

c m : per haem at 428 rim. All experiments ~ere carried out at 20°(" and in 5tl

mM phosphate buffer (pH 7.2). H,O~ ~dution.~ of i0 mM !ab~rbance coefficient 43.6 M -~ .cm : at 240 nm,~ were prepared freshl3 from a 30~ >t~'k ~olut~on (Merck). 5-Aminosalicylic acid was obtained from Merck. All other chemicals ~l.-~d ~,ere of the highest purity available.

The chlonnating activity o f m~eloperoxidase ~a~ dr- terrmned using monochlortxtimedone as a ,~avenger for hypochiorous acid [7]. The monochlorodimedonc ab- sorption d e c r e ~ was measured at 290 nm (19.9 mM- -cm ~) wid~ the use of an Union-Giken RA-401 stopped-flow spectrophotometer. The time intervals used were 0-200 ms, 0-1000 ms and 0-60 s. From the slope of the trace, the number of converted monochloro- dimedone moleculo per second per haem group of myeloperoxadase (turnover number) was determined over several time intervals (see figures in Results). For the determination of the Km for H_,O_,, three different plot methods were used. namely the activity versu..;

]41

sub>Irate concentration p!ot. the Linesveavcr-Burk plot and the Eadie-Hofstee plot. Transient spectra of myeloperoxida~ were taken with the Union-Giken RA- 401 rapid-scan spoctrophotometer covenng ,~: wave- length range from 402 nm to 498 rim.

Resul ts

Formation of hypochlorous acid by myeiol~rc,xidase can only be meast~red indirectly using a compound that changes absorbance upon chlorination, such a~ mono- chlorodimedone [1 i ].

In Fig. 1 the chlorinating activity of myeloperoxidase is plotted against the H:O; concentration at pH 7.2. With 100 pM H~O2, the turnover number reached maximally was about 320 s ~ p e r haem in the first 100 m,~ and thereafter the activity decreased to abou;. 25 s per hae'n at 1 s after nuxin~ and ,o about 13 s - ' per haem (not shown) at 15 s. At Io~er concentratiop.s of H:O_, ( < 50 /~ML the turnover rates were less depen- dent on time. These results are in good agreement with the suggestion [20.221 thai the lower activity of m)elcq3eroxidase at neutral pH values is mainly a result of the reaction of Compound ! with H20_,. producing inactive Compound 11. Monochlorodimedone had a small contributior to the formation of Compound Ii as will be described later. Because the inactivation of myeloperoxid.~se was dependent upon both time and t i ,O: concemra,ion (Fig. I ), the apparent K m for H zO_, became ai.~ depetldent on time. For the time interval with the highest actiwty, namely from 0 to 100 ms, a K,. of al~ut ~[) tLM was calculated.

'7 t/'?,

Z

j ....

320 ~ J

i / i /

2~,0 t ,o" 4, /

e !

If~0 ~" £ . . . . . . . . . . . . ~ - .... ' / / • t

.... b . . . . L~

C t.8 ~ 120 ~60 200

I H202 1 I ~M

Fig, I *~ch~.it', o l rn~ .eh~eroxJda~ . J.~ turrK)~,cr numl"~r~ s -~ po"

haem. a~ a [um:tJon ~ff H , O : con~ :n t ra t Jon . Lin~. are d r a ~ n f~ r

~anou~ t im( mtcr~al~ after a d d m , m of H : O z (start of the reacuoQ):

0-1013 rn~ (~): I00- ~JO m.~ (~) and 750-I00) ms (~). The re_~ction reed,urn conlamo:h 40 n.M m)cloperoxldase. 206 mM KCL 50 pM

m,)~<hlorcvJJmo.~.: and ~ mM pho,4~hate buffer {pH 7.2).

142

"5-

Z I . -

320

2~0

160

8 0 ¸

o i

o i

/ e,

,

- I

_ _ 1 0 ~0 80 ~20 W_)O 200

1H2021 ~ )

Fig. 2. Act0.'tl~ of myelopero,tida~, a~ turnover numbers s ~ per haem. in the presence of l0/aNt 5-amino.,,ahcyli¢ acid as a fun~tiota of H zO: concentration. Lines arc drawn for various time inte~'als after addition of H:O~ Istdrt of t~e reaction): 0-100 ms (o); ICO-200 ms

(z~); 500-1000 ms(U). Reaction medmm as m the le-aend to Fig. I.

When experiments were carried out tn the presence of the reductant 5-aminosalicylic acid, however, the turnover numbers at various times and H 2 0 2 concentra- tions were different, as is shown in Fig. 2. The initial 'maximum" vekx'ity was slightly lowered ba t on a longer time scale (100-1000 ms) the rate remained at the much higher level of about 150 s- ~ per haem as compared to 25 s - I per haem in the absence of 5-aminosalicylic acid. This turnover rate was stable until about 5 s, af ter which a decrease in activity occurred as a result of consumption of H , O , to rate-l imit ing concentra t ions (not show, l . In the pre:~nce of 5-aminosalicylic acid. a value of about 30 p M for the K m for H,O2 was found during the time inter~'als used ( < 1000 ms).

To determine whether 5-aminosalicylic acid pre- vented inactivation of myelopero. 'ddase by inhibi t ing the formation of inactive Compound 11 or by con~er- sion of Compound II back to native enzyme, we first :~ad to c,,,Aude the possibi!ity that 5-aminosalicylic acid reacted with moncxhlorodimedone itself. It was found that 5-amlnosalicylic acid did not react with monochlo- ro, dimedone, not even in the presence of H , O , (not shown). This enabled us to per form exper iments in w-hich the monochlorodimedone absorpt ion decreases at 290 nm resulting from myeloperoxidase activity were recorded as a function of t ime (0-11300 ms) in the presence and absence of 5-aminosalicylic acid a n d / o r ascorbic acid (Fig. 3). The myeloperoxidase concentra- tion u~ed (2.6 vtM) was much higher than in the experi- ment~ de~'r ib~d above ~40 nM), so it was possible to follow the transient myelopertxudase spectra as well under the same condit ions. Fig. 3 sho~s that, under the condit ions used. the reductant 5-aminos,dic,,l ic acid had a stim.ulating effect on the activit,, r of myeloperoxidase.

which was much grca~er compared to that of ascorbic acid especial ly after about 200 ms. At a r e D short t ime interval (0 -25 ms) these reductants rather had an in- hibi tory effect than a s t imulat ing effect. This is prob- abl) ' due to react ion of C o m p o u n d I with these re- duc tan ts to C o m p o u n d II. leading to an initially lower s teady-s ta te level of C o m p o u n d I and lower chlor inat - ing activity.

As can be j u d g e d from Fig. 4A, C, ' ,mpound l], which absorbs at 456 nm. was actual ly formed, and format ion was near ly comple te within 352 ms. As a result of C o m p o u n d !1 format ion, the enzymic activity becanm very. low (cf. Fig. 3). When 5-aminosalicylic acid was

present. Compound I! was also produced, but a rapid conversion to native enzyme ~,hich absorbs at 428 nm

took place, as is shown in Fig. 4B. I,I contrast to

5-aminosalicylic acid. ascorbic acid caused no con-

version of the produced Compound il to native myeloperoxidase m the time range studied (F,~,. 4CL

The posiuon of the isosbestic points at 440-445 ran.

compared to that reported in the literature [28]. showed

that in the presence of either of the two reductants the

Compound formed was indeed Compound II. IP.. the absence of reducmnts a shifl was noticed in the isosbes-

tic points during the e~periment. It was concluded that

in this case also some Compound III was formed in

addition to Compound II.

To confirm that 5-aminosalicylic acid was more ef- fective as an electron donor in the conversion of Com-

pound I! to native enzyme than ascorbic acid. the rate constants for this reaction were determined directly.

Compound [I was mixed at pH 7.2 with different con- centrations of the electron donors and pseudo-tint-order

rate constants were calculated for the decay of Corn-

%%

250

02

4.

i i

500 750 1000 t (ms)

Ftg. 3. Time conr~e of t l~ absorptton decreases at 290 nm ~ a rcsuh of chlonnatton <.i monochlorodimedone carMvsed by myelopevox.t-

~'ta its product hypochloro~5 actd. The , ' ~ t t on me.urn tamed: 2.6 ~M mydo~rox/das~. 50 mM KCL 150 ~ M mom:~do¢o~ me.one and 50 mM phosphate I~Jff~ tpH 7.2) in the aloe'nee (--) or pr,~m,--e ~ ...... ) or 50 pM ascod~ a~d or 50 ~M 5-armu~sabc)bc acid ( ..... ). The reaction was ~tarted by a~Ittaoa

of 150 jt,S.I H:O:.

0 2

oi

! d

~02 ~ ~ 5 0 ~75 ~ 9 8

~2

01

0 t.02 /25 t.50 t.75 ~98

i n to )

02 / /

°' i i

0 , , , I 1.02 ~.25 : 5 0 ~.75 ~98

ig. 4. Aly~r lp l~r l ~ t r a o f m~,z~itcroxida-s¢, unde r the turr~_~,cr mdi~.mo.s dles~rib¢~l in F i g 3, a l ~, "~Ot~$ lit 'l~ int¢'~aL', a | t c r ~ l ~ l | l o n

150 ~ M H : O = (s tar t o f the rc~=uon), l a the a b ~ n c ¢ (A~ o~ re~cta.-¢ o f 50 ~ M 5-aattr~-,sahc'yhc acid ~B) of" 50 ~ M a.~ca 'b . : ..~.~d 7). Titr~ mterxrals u:~d ~.,en¢: {in A) 44-46 m.~ (a); g8-90 ms (h~: 76-1"/8 ms (eL 3 5 2 - 3 5 4 ms amid 9 5 2 - ~ 6 2 ms (d). (m B) 112 ..122 ms

O: 2 3 2 - 2 4 2 ms (b); 3 5 2 - 3 6 2 ms Ic); 4 7 2 - 4 8 2 ms Id~. 5 9 2 - 6 0 2 m ,

t ; 712 -T22 ms (f): 8 3 2 - 8 4 2 ms (g); a n d 9 5 2 - 9 6 2 ms ~hi. tm ( ' )

I . -46 ms (a): 8 8 - 9 0 ms (b): 176--178 ms ~c) 3 5 2 - 3 M ms (d) and

9 5 2 - 9 6 2 ms (d).

143

pound I!. These rate constants were linearly depende~,t upon the concent raucn of 5-aminosalicyhc acid and ascorbic acid (not shown). The second-order rate con- stants for the conversion of ~'ompound ll to native enzyme as calculated from the slope of the lines, were 3.2 • 1 0 ~ M i . s - i for 5-amino$tlicylic acid and !.5 - 104 M - n. s- 1 for a~o rb i c acid. rt~pectivel),. 5-Aminosali- cylic acid and ascorbic acid dic! not affect the spectrum of C o m p o u n d I l l (not shown in f igure) .

Kettle - ~ a,,~ Win te rboum ' " " t'~"~ had shown that at pH 7.8 monochlorodimedon¢ could react with Compound !

to yield C o m p o u n d il. Un.:ler our assay conditions (pH 7.2) this reaction would i~rohably occur also. To in- ve.qigate this possibility the enzymic activity of myeloperoxidase was measured as a function o f the monochlorodimedone concentration from I0 to 100/~M under different conditions. Different concentrm.ions of monochlorodimmJ_,~na only sliehtlv affected the acti , i tv of myeloper: ,~dase dunng the time interval 10-100 ms and 500-1000 ms. as is shown in Fig. 5A. However. myelopcroxidas¢ became inactivated somewhat earlier at higher monochlorodimedonc concentrations, which shows tha, besides H20 , also monochlorodimedone contributed to the Compound II formation (nn: shown in F i g . 5 A ) . In Fng. 5B the effect of mop.ochloro- d imedone concentrat ion is given in the pr~'-,ence of 5-aminosalicylic acid. Here the absorption decreases at 290 nm of (different concentrations of) monochloro- d~medone are plotted against dine (0-1000 ms). The turnover numbers. *hich are the negative slopes of the traces, were not slgnificant!y affected by yawing the m(,flochlorodimedone concentration.

In Fig. 6 the turnover of myeloperoxidase is shown as a function of the chloride concentration. Contrar) ' to the obserx'ation~ v.ith H . O : (F~. l). where the decrease in rate as a function of tm'~ ,~'as more pronounced at relati~eh high concentrations of H . O , . in the presence of low chloride concentratn,gns the turnover rate de- , . rca~d .,~rongl~ ~ith time. When high ~:oncentra,ions of chloride ge re present the turnover rates ~¢re far le~s dependent on time. This sugge~,rs that at high chloride concentrat :ons the~e ~as much iess accumulation of Compound I!. This effect can be explained by competi- ti6n of chloride and H ,O, ~r mGnochlorodnmedone for reaction with Compound i. Upon reaction of Com- pound i v,lth chloride native enz)me will be produced.

hereas with H .O. or rmmochlorc~tmedone Compound II formation will occur [22].

High concentrauons o[ eMonde also had another effect on the Compound !i formation: the amount decrea,,cd ~ a r~ui t of complex formation bet,,~een cMoride and protonated natise m)eloperoxidas¢ 117.20] to ) leid an matinee m)doperoxlda~e-chlonde complex. In Fig. 6 inlubmon b,, chloride is ~isible at chloride concentration~ abo~e 200 raM. particularly dunng the first 50 ms of the rcactmn Chloride inhibition ~.as

144

" i 27

~+" - ~ .;'72~27 -~+~. .___x+-__-_. --°27~-+'r--+-"----- t~T ++ ' -91~

O 2C ~0 50 80 ~00

~CDI (uM)

Fig 5. ~X).+Xct~+t~, of ms'elopero~da,e, as turnover aumbers s ;

I

* i

[

_ I

; 250 500 750 ~000 ' : t ' r ' $ I

Ixr haem. a~ a fanctlon of n'~.)n~xhlortKh~¢ ¢onct:ntration. The r~..aetioe medium contain,M: .V.) nM m?,el,~pero~,da.~. 2{)0 mM KCI. _~0 mM pho~,phate buffer tpH 7 2) and nx.'mochk-rodimcdon¢ { ~ figure}. The re.a~tlon v,'a~; ~,tarted by addition of 100 pN/. H202. "File time lnter~'als of the measurements u,¢l¢ 0-100 ms ¢~) a.,~l .~00-1000 ms {r._q). tBj Ab.~wptton decrea~ ~, a: 290 nm as the re~;u|t of chlorination of monc~:hlort~llmedone in the pre~'r~ce of 5-aminosahcyh¢ acid plotted against time {0-1000 ms.)_ '!he negau~e slope~ of the traces represent the turnover numtmrs. The fotk~,vlng co.,~entratttms of mone~hlot'edimedor.e wt"re used: l0 pM (a); 20 ~gl IbL 35 ~M re): 50 pM (d): 65 pM (e): 80 oM (f): 100 ~tM (gJ. The reactitm medium contained: 40 nM myelop~o~Jdase. ~ mM KCL 50 mM

phosphate buffer {pH 7.2). l0 pM 5-amtnt~,alic',h¢ acid and rmmt~hlort~ltmoJon¢. ~ reaction wa~ started by additkm of 100 pM H202

hardl> oh~r~ed after 150-500 ms. This shows that the chlor tde-myeloperoxidase complex was d issoc ia ted within 150 500 ms afte, mixing with H , O , . This effect of chloride appeared to b,: independent of the presence or absence of 5-aminosalicylic acid or ascorbic acid. Because of these two described chlor ide effects, efforts to determine the Km for chloride were not successful. By comparing Figs. 6A, B and C it is clear again that the ab{htv o f 5-aminosalicylic acid to diminish accumu- !ation of Compound II was grcat::r than that of a~ 'orb ic acid. but as expected both were most effective on the my¢loperoxidase activity when the chloride concentra- lion wa, relatively I0~.

D i s c u s s i o n

Tht' enzxmic actixitx of m>eloperox ida~ is usually deteram~'d after mixing with H . O . from I0 .,, onwards u~ing ~t ~:onvcntional spectrophotometer . As shown in the prc~cnt paper, at pH 7.2 near[~ all native m~eloperoxida~e was rapidly conxerted to C o m p o u n d ll within 10 ~. especiall~ at high | t : O , and low chloride concentrations. That accumulat ion of Compound II account.~ for the docrea:~ in enzymic activity at neutral pH values was previously suggested by other authors [20.21.23.29-31]. [:or this reason studying the kinetic pard.meters of myeloperoxidase at neutral p H is ve~. compli~:,tted and hardly practicable. In previous papers we showed that ascorbic acid "activates" mye lo lx rox id - a~e or, more pt~'c~s¢ly, regenerates native enzyme from Compound II [23.25}. in the premnt paper we den~sn- strafe that. even in the presence of a.~orbic acid. still a significant amount of Compound l l accumulated. In the presence of 5-aminosalicylic acid. also a considerable amoum of Comixmnd l l was formed initially, but con-

version of C o m p o u n d .~1 "o native enzyme was strongly accelerated by this reductant. Thus after 50-100 ms, the turnover rate in tl, e presence of 5-aminosal icycl ic acid became much fa~ter than in its abse/~ce, i t has been shown [24,32} that monoch io rod imedone is able to con- vert mye loperox idase to C o m p o u n d !1. In view of this it m a ) be expected that monoch lo rod imedone affects the enzymic activi ty in our as .~y system. However, under our experirncnt.,d condi t ions , no gross effect of mono- ch lorodimedon¢ was observed. Fur thermore . the small con t r ibu t ion of monochlor tx t imedone to the C ' . m p o u n d 11 format ion under our assay condi t ions does not affect our conclusions concernin$ high initial act ivi ty of myeloperoxida~: and act ivat ion by electron donors such as 5-aminosahc>lic acid. Unfor tunate ly . there are n o

known al ternat ives for measur ing the hypochiorous acid p roduc t ion of mye loperox idase without any side effect{s).

Our exper iments show that the enzymic activi ty of m~,eloperoxidase is s trongly dependen t on the experi- mental condi t ions , in agreement with earl ier work [14.18,20,21.23.25.31.33]. Not only substra te concentra- tions (H=O: and chloride) and p H values are of impor- tance, but the t ime interx, al of the measurement , the presence and concent ra t ions of reductants and con- centra t ion of myeloperoxidase also p lay a role. A n addi t ional diff icul ty is the fact that bo th substrates ( H : O , and ch lor ide) a lso act as inh ib i tors o f myeloperoxidase t17]. With these l imita t ions s teady-s ta te kinetic analysis of myeloperoxidase is complicated. At each p H value it is n e ~ to deternun¢ the op t imal condition~. Titis palx:t ~ht,~s that ,it neutrai p H an effort to de termine the K, , for H , O , and the "maxi- mum" velocity of myeloperomdase is only just i f ied using the t ime interx-al 0 - 1 0 0 ms after addi t ion of HzO z to the

320

320

160

|

z

160

B

320 ' ]

160

0 2 ~ 5 ~ 7 ~ 1000

I K C t l traM)

Fig. 6. Ac:-,~ly of m~.ck~rox~d.c~¢, a.~ turno,.er number,, • ~ per a.', a fumt£ot+ : , i th~ chk~le ct3r~enltalton Lxn~ arc dra'~,n for

vano~t&s tzro¢ tnt~r~als afl~ ~:]ditncm of lCW~ ~, t H.O- ~•ta~ ,ff the r¢.act~0n): O-.C,0 ms ( : ' ) : I.C,0-.N)O ms (,.~): ~ 0 m , ( ~ I a n d " ~ ) lt~'W~ ms 1~2). Th~ rtm~=t~on m e d i u m ct.~ta~lx 'd: 40 nX| rn>¢!,,Ix'to~!da,~.-, .~+~

~.M flt~o.l~hk,~rodlm~wdolt~ KCI I~-~ [lgl.lr¢l .:itqd .~0 reX| pho: ,phat¢

buf fe r I p H ~2.~ m the ab~ '~ce ~ A) ,~ presen~z o[ 10 a%t a-,corbt¢ a,:td (Be of 10 ~tXI 5-an~no-~ahc-,hc acid I(-~.

reaction medium. When longer time interval.,, were used. 5-azmm~alic~'ltc acid had to be present in the reaction medium to k~-~-p the amount of Compound il lo~. thu.,, maintaining the enzymic acridly of myeioperoxidase at a constant level. Using the above-m.attioned conditions in the absence and presence of 5-aminomliojhc acid a reasonable estimate of the Km for H:O: at pH 7.2 and 200 mM chloride is about 30 ~aM. This K~, value ig in line ~ilh the results of Bakkemst ¢t al. {20] and Zgliczyhski ea at. [34] who showed that the logarithm of the K= for H.O~. decreased linearly with pH. Extrapola- tion of th¢ data in Ref. 33 to pH 7 yields a value of about 10/tM for the K m at 200 mM chloride. Wever et aL [31] estimated a "true" K= for H .O: of 32 pM at a pH value higher than 6 on the basis of steady-state

145

experiments. Thus our value is in good agreement with th]~ work. Unfortunately. a Km for chloride cannot be given yet. becau~ in case of this substrate inhibition is present which interferes with the determination of the Km. However. on the basis of our data. the K,. for chloride was in the millimolar range (Fig. 6(').

The conditions of the environment which are encountered by myeioperoxidase in vivo are still a point of di~xtssion. Earlier investigations [35-37] suggested an acid pH. from 4-6. This was in agreement with the relatively high activity of myeloperoxida`s¢ measured at these pH values [20.28]. Recent evidem:e, however, sug- gests that the pH value in the phagosom¢ just after the initiation of phagocytosis is neutral [26.38]. Thus. it is relevant to know whether myeloperoxidase is catalyti- cally active at neutral pH. In particular since it was observed ~i th conventional assay methods thai myeloperoxidase has a ve~' low acti,ity at neutral pH. From the p re~n t paper it is cl~-r that myeloperoxidase has a much higher activity a~ neutral pH during the first 100 to 200 ms after c('ntact with H,O, than at later. usually measured, time intervals in which the rate de- creases to about 5~ of the initial rate. When wc assume that the same holds for the situation in vivo we have to conclude that just after the first contact with H : O 2 a burst of hyix)chiorous acid is produced to attack the nu~ro-organism(s). Dunng this first contact period myeloperoxida~ probably acts under optimal condi- tions. This is suplx~rted by the low value of the K~ for H:O: of about 30 pM (pr:sent paper) at neutral pH as compared to the K~ value of 7.7 mM at pH 4.4 [18]. The inactivation of myeloperoxidasc depends on the competition of H,O. and chloride ions for Compound !. Therefore the "steady-state" concentration of H.,O:. determined by its generation via the "oxidative burst" and consumption of myeloperoxidas¢, is crucial. How- ever. the ctmcentratton of H , O , in the nn ~,ivo situation is not known. Considering the [or. K~, value for H20 - . it is likely that we d¢~.l with a stead',-state concentration of H_.O: v,hich is relatively Io~,.

Concerning the second ',ubstrate chloride, the condi- tions for myeioperoxidas¢ m vb.o are aim<r.! optimal, as deduced from the fact that at the chloride concentration in the phagt~,ome. ,,,,hich u.,, about I(K) mM [36.39]. myeloperoxidas¢ had a relau~ely high activity (Fig. 6). After a period in which myeloperoxidase probably func- tions with a high actbity, the enzyme becan~ in- activated by its own sub, irate H:O~ due to formation of Compound II. From this point of view H:O: has a regulato~ function by inhibiting the chlorinating activ- ity' of myelopero~udase In the pre~ent paper it is sho~n. however, that other compounds, such as ascorbic acid and 5-anunosalic~ltc acid. have a stimulating instead of an inhibiting effect on the .-.ctivit~' of myelopero~da.s¢. In this respect it is of inter~t to note that superoxid¢ ~O,) is also able to regenerate active m~eioperoxidase

146

from Compound II [40]. In particular the stimulation of the activity ol myeloperoxidase by ascorbic acid is probably of physiological importance. ,tr:ce human neu- troptuls are knov, n to contain ascorbic acid [,11]. Hog'- ever. it remains to be established that this reductant has a regulator, role in the activity of myeloperoxidase.

Acknowledgements

We wish to thank Professor B.F. van Gelder and Dr. H. Hoogland for critical comment and fruitful discus- ~ion. We thank H.L. Dekker for skillful technical assis- tance during the initial phase of this investigation. This study was supported in part by grants from the Nether- lands League against Rheumatism, and in part by grants from the Netherlands Organization for Scientific Re- search (NWO) under the auspice~ of the Netherlands Foundation for Chemical Research (SON).

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