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 . B a k k e n i s t , R. W e v e r a n d A . O . M u i j s e 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 II; 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 polymorphonuclear leukocyte [1-4 I. After phagocytosis, myeloperoxidase is excreted into the phagosome [2]. From chloride. which is always present, and H_,O:, which is generated 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 myeloperoxidave 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 Compound Ii [21-22], which is an inactive form of myeloperoxidase with regard to formation of hypochlorous acid [15,23]. Because of this progressive inactivation 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 myeloperoxidase 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 catalytic reaction b~ reducing Comv~,und I to Compound
(,1C74~3' ~).'$U3.SLI ' I~h) Eb,e~,er ."~.mnce Publishers B ~ (~honledllca! L~,,1,qon)
]41
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 measurement methods, the activity of myeioperoxidase in the presence of ascorbic acid decreased in time and accurate _ae.lermination of kinetic parameters, such as maximal 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 a b ~ n c e and presence ol ascorbic acid. usir.g a stopl:~d-flow/rapid-scan ~pectrophotometer. 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 myeloperoxidase kinetit,~ were a l ~ studied in the pre~nce of 5aminosalicylic acid. Under well-defined conditions it was possible to determine a K,, for H_.O:. M a l e r i a i s and M e t h o ~
Myeloperoxidase was purified from h u m a n leukocytes, following the method d e ~ - r i b e d in Ref. 27. Myeloperoxidase preparations with a ,;28 n m / 2 8 0 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 ! a b ~ r b a n c e coefficient 43.6 M - ~ . c m : 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~ drterrmned using monochlortxtimedone as a ,~avenger for hypochiorous acid [7]. The monochlorodimedonc absorption d e c r e ~ was measured at 290 nm (19.9 m M -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 - 6 0 s. From the slope of the trace, the number of converted monochlorodimedone 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..;
sub>Irate concentration p!ot. the Linesveavcr-Burk plot and the Eadie-Hofstee plot. Transient spectra of myeloperoxida~ were taken with the Union-Giken RA401 rapid-scan spoctrophotometer covenng ,~: wavelength range from 402 nm to 498 rim. Results
Formation of hypochlorous acid by myeiol~rc,xidase can only be meast~red indirectly using a compound that changes absorbance upon chlorination, such a~ monochlorodimedone [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 dependent 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.
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Fig, I *~ch~.it', o l r n ~ . e h ~ e r o x J d a ~ . J.~ turrK)~,cr n u m l " ~ r ~ s - ~ h a e m . a~ a [um:tJon ~ff H , O : c o n ~ : n t r a t J o n . Lin~. are d r a ~ n
po" f~r
~ a n o u ~ t i m ( 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,)~
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 . B a k k e n i s t , R. W e v e r a n d A . O . M u i j s e 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 II; 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 polymorphonuclear leukocyte [1-4 I. After phagocytosis, myeloperoxidase is excreted into the phagosome [2]. From chloride. which is always present, and H_,O:, which is generated 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 myeloperoxidave 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 Compound Ii [21-22], which is an inactive form of myeloperoxidase with regard to formation of hypochlorous acid [15,23]. Because of this progressive inactivation 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 myeloperoxidase 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 catalytic reaction b~ reducing Comv~,und I to Compound
(,1C74~3' ~).'$U3.SLI ' I~h) Eb,e~,er ."~.mnce Publishers B ~ (~honledllca! L~,,1,qon)
]41
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 measurement methods, the activity of myeioperoxidase in the presence of ascorbic acid decreased in time and accurate _ae.lermination of kinetic parameters, such as maximal 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 a b ~ n c e and presence ol ascorbic acid. usir.g a stopl:~d-flow/rapid-scan ~pectrophotometer. 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 myeloperoxidase kinetit,~ were a l ~ studied in the pre~nce of 5aminosalicylic acid. Under well-defined conditions it was possible to determine a K,, for H_.O:. M a l e r i a i s and M e t h o ~
Myeloperoxidase was purified from h u m a n leukocytes, following the method d e ~ - r i b e d in Ref. 27. Myeloperoxidase preparations with a ,;28 n m / 2 8 0 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 ! a b ~ r b a n c e coefficient 43.6 M - ~ . c m : 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~ drterrmned using monochlortxtimedone as a ,~avenger for hypochiorous acid [7]. The monochlorodimedonc absorption d e c r e ~ was measured at 290 nm (19.9 m M -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 - 6 0 s. From the slope of the trace, the number of converted monochlorodimedone 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..;
sub>Irate concentration p!ot. the Linesveavcr-Burk plot and the Eadie-Hofstee plot. Transient spectra of myeloperoxida~ were taken with the Union-Giken RA401 rapid-scan spoctrophotometer covenng ,~: wavelength range from 402 nm to 498 rim. Results
Formation of hypochlorous acid by myeiol~rc,xidase can only be meast~red indirectly using a compound that changes absorbance upon chlorination, such a~ monochlorodimedone [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 dependent 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.
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Fig, I *~ch~.it', o l r n ~ . e h ~ e r o x J d a ~ . J.~ turrK)~,cr n u m l " ~ r ~ s - ~ h a e m . a~ a [um:tJon ~ff H , O : c o n ~ : n t r a t J o n . Lin~. are d r a ~ n
po" f~r
~ a n o u ~ t i m ( 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,)~
