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Effect of 2,3-butanedione on human myeloperoxidase
lirr. 2. Bioehem. Vol.21, No. 7, pp. 735-7759, 1989 Printed in Great Britain. All rightsreserved
0020-71 IX/S9 $3.00 + 0.00 Copyright 0 1989 Pcrgamon Press plc
EFFECT OF 2,3-B~ANEDIONE ON HUMAN MYELOPEROXIDASE Nus PRDERWILLASSEN and CLIVE LITTLE Institute of Medical Biology, University of Tromse, P.O. Box. 977, N-!X#l Tromsr& Norway [Tel (083) 86560] (Received 15 November 1988)
Abstract-1. Myeloperoxidase is inhibited by various diketones that are recognized arginine reagents. 2. Although arginine residues in the enzyme were modified in both the light and the dark, enzyme inactivation occurred oniy in the presence of light. 3. Under conditions where diketones caused inactivation of myelo~ro~~, spectral studies indicated
marked damage to the haem residues of the enxyme. 4. It was concluded that diketones serve simply as photosensitizers of visible light-induced inactivation of myeloperoxidase. 5. Studies on other haemoproteins indicated the great ease with which the presence of diketones sensitized haem residues for photodestrnction.
Methods
IN’IXODUCHON
isolation and assay of enzyme
Myeloperoxidase constitutes 2-5% of dry wt of neutrophils (Schultz and Kaminker, 1962; Nauseef er of., 1983) and appears to have anti~~obi~dal functions (Klebanoff, 1975; Clark, 1983). The enxyme has a molecular weight around 140,000 (Ehrenberg and Agner, 1958) and contains two green haem prosthetic groups (Agner, 1958). The normal human enzyme seems to be a tetramer comprising two identical large (M~57,~) and two identical small (M, 10,500) subunits (Olsen and Little, 1984). The haem residues, which seem associated with the large subunit (Harrison et al., 1977; Atkin et al., 1982) are essential for the catalytic function. Little, however, is known regarding the possible participation of apoprotein in substrate binding and catalytic activity. Prompted by the control role of arginine in the proposed m~hanism of action of cytochrome c peroxidase (Poulos and Kraut, 1980a,b), we decided to investigate the effect of certain diketones on myeloperoxidase. Such reagents are commonly used for probing for “essential” arginine residues in proteins [see Aurebekk and Little (1977) and the review by Lundblad and Noyes (1984)].
Myeloperoxidase was purified from butTy coats of outdated human blood as described by Olsen and Little (1983). Myeloperoxidase with a A,r,r,fAzsor_ ratio (Rz value) between 0.81 and 0.83 was used. Enzyme of this grade of purity can be crystallii (Sutton e; a/., 1988). -Enzyme activity measurements were based on the method of Shindler et al. (1976). Assays were carried out in a 50mM sodium acetate buffer, pH 4.4, with fmal concentrations of hydrogen peroxide and 2,~-~n~i-(3~hyl~~oline~s~phonic acid) of 0.05 and I mM, respectively. The reaction was followed continuously at 412nm in a Hitachi 557 double-beam recording spectrophotometer at 22-24°C. Chemical modification reactions inactivation by l,.kyclohexanedione. Treatment of myeloperoxidase with 1,2-cyclohexanedionewas carried out at 37°C in a 2 ml reaction mixture containing myeloperoxidase (0.25 mg/ml) and 1~rnM 1,2~yclohexan~ione in
MATERIALSANDMETHODS Materials
2,2’-Azinobis(3-ethyIbenzothiazoline-6-sulfphonic acid) was purchased from Boehringer Mannheim GmbH (F.R.G.). N-Bromos~~mide was obtained from J. T. Baker (Deventer, The Ne~erlands). ~io~ycoltic acid, 2,3butanedione (freshly distilled before use), 1,2-cyclohexanedione (recrystallized) and phenylgfyoxal were from Ruka AG (Switzerland). Lactoneroxidase, horseradish neroxidase and ‘cytochrome c were from Sigma (St. Louis, MO. U.S.A.). H,O, was obtained from the local chemist as a 30% (w/v) SbluGon. All other chemicals were of analytical grade purity.
50 mM sodium borate buffer, pH 8.0. During the course of reaction aliquots were removed, diluted with the incubation buffer at room temperature (22-24”C), and assayed for remaining enxyme activity. Myeloperoxidase incubated with buffer alone served as control. Zrzactivation &y ~ny~giyoxa~. A 0.5ml solution of myelo~ro~d~ (0.5 m&ml) in 100 mM sodium bicarbonate buffer, pH 9.0 was treated by adding a 0.5 ml solution containing different concentrations of phenylglyoxal in the same buffer. Myeloperoxidase incubated with butfer alone served as a control. Aliquots were removed from both the sample and control, diluted with the incubation buffer and immediately assayed for enzyme activity. All reactions were carried out at room temperature. I~t~va~ion by .2,3-butanedione. Aliquots from a pHadjusted 2,3-butane&one-borate solution were added to myeloperoxidase (0.25- 1.Omg/ml) in 50 mM sodium borate buffer, pH 8.0 to initiate the modification reaction. This procedure was followed unless otherwise stated. Enzyme incubated with buffer alone served as untreated control. To stop the reaction, samples (IO-25~1) were withdrawn from the reaction mixture at different time periods and
755
756
NIB
PEDER
WILLASSEN and CLIVELITTLE
diluted with the incubation buffer. The enzyme activity was measured immediately afterwards. The modification was performed at room temperature. Ihmination
of samples during inactivation
Unless otherwise stated, incubation were performed in a room illuminated only by normal fluorescent light tubes. The light intensity on the reaction mixtures was measured using a HIOKI Lux Hi Tester 342 1. Amino acid analysis
Protein samples were precipitated with 20% (v/v) trichloroacetic acid (TCA), cooled and centrifuged. The precipitated proteins were washed with 6 M HCl to remove excess modification reagent. The proteins were hydrolysed for 24 hr in 6 M HCl containing 0.05% (v/v) thioglycollic acid at 110°C in glass tubes sealed under nitrogen flushing. After hydrolysis the sample were dried in a vacuum oven and dissolved directly in a 0.2 M sodium citrate buffer, pH 2.2, containing 0.5% (v/v) thiodiglycol. The quantitative amino acid analysis was performed in a JEOL JLC-6 AH amino acid analyser. Tryptophan was determined by using N-bromosuccinimide, essentially as described by Spade and Witkop (1967), on myeloperoxidase dissolved in 6 M guanidinium chloride. RESULTS
Inactivation by arginine reagents Myeloperoxidase was exposed to three different standard arginine reagents, viz. phenylglyoxal, 1,2cyclohexanedione and 2,3-butanedione. All three were able to inactivate the enzyme. Under the reaction conditions used, 2,3-butanedione inactivated the enzyme in a classic pseudo-first order kinetic manner, whereas the kinetics of inactivation by the other two reagents were more complex (Fig. 1). The inactivation by 2,3-butanedione was investigated further. In cases of pseudo-first order inactivation, the slope of plots of log [pseudo-first order rate constant] vs
10’ 0
o . n 10 20
n
c
40
30
’
SO
,.4
1.6
,.3
2.0
2.2
Time (mln)
log [reagent concentration] indicate the number of molecules of reagent reacting to inactivate one active centre in the enzyme (Levy et al., 1963). Applying such an analysis to the data for the inactivation of myeloperoxidase by 2,3-butanedione yielded a straight line of 0.72 (Fig. 2), thereby indicating that no more than one molecule of reagent was needed to inactivate each active centre in the enzyme. Nature of the chemical modiJication causing enzyme inactivation Myeloperoxidase is characterized by possessing a strong green colour. During incubation with an inactivation by 2,3-butanedione a visible bleaching of the enzyme occurred. This effect was studied more closely. Spectral studies carried out every 30min
2.4
Log(I2,3-butmadbn.]rlOOO)
IO 0
I
I
I
10
20
30
Time
Fig. 2. Inactivation of 22-24°C by treatment was incubated in light (O), 100 mM (m) and
’
Fig. 1. The rate of inactivation of myeloperoxidase with different arginine modifying reagents. Myeloperoxidase (0.25 mg/ml) was incubated in visible light (400 lx) with 100 mM 1,2_cyclohexanedione (A), 50 mM phenylglyoxal (a) and IOOmM 2,3-butanedione (0) as described in the “Materials and Methods” section.
100
1.2
’
60
40
I
I
50
60
(min)
myeloperoxidase by 2,3-butane&one. The enzyme (0.25 mg/ml) was inactivated at with 2,3-butanedione as described in “Materials and Methods”. Myeloperoxidase (400 lx) with the following concentrations of 2,3-butanedione; 25 mM (A), 50 mM 200 mM (0) in 50 mM sodium borate buffer, pH 8.0. Inset: plot of log (pseudo-first order inactivation) vs log (reagent concentration).
151
Inactivation of myeloperoxidase 0.6
I.16
0.6
I.10 0) ::
8
zi
p”
,o
9
f
5
0.4
Q
0.2
0””
Z””
4””
Wavelength
El””
0””
(nm)
Fig. 3. Effect of 2,3-butanedione on U.V. and visible spectra of myeloperoxidase. The enzyme (2 ml, OJmg/ml) in a quarts cuvette was incubated with 3~1 2,3-butanedione (17mM) and irradiated with visible light (260 lx). The spectra were measured, every 30 min for 8 hr. The ordinate on the left is from 200 to 500 nm, and the right from 500 to 640 nm. The absorption values decreased progressively during the experiment. during the reaction showed a marked diminution in the absorbance bands characteristic of the haem residue and a smaller effect on the tyrosine/ tryptophan absorbance around 280 mn was also observed (Fig. 3). Both the inactivation and the spectral changes were absent if the sample was protected from light. The changes in enzymatic activity and Rz value &,,/A& during the incubation with 2,3-butanedione in light or protected from light are shown Fig. 4. In the dark, a small, but completely reproducible activation (cu. 10%) of the enzyme
- 1.0 - 0.8
occurred. From an extrapolation of a replot of the data in the form of percentage inactivation against percentage loss of absorbance at 430 or 280 nm (not shown), it was estimated that the total inactivation (in light) corresponded to a 50% decrease in the absorbance due to the haem residues and about 20% at 280nm. In order to test for the modification of specific amino acid residues, the amino acid composition of native and 2,3-butanedione-treated (in the light and dark) enzyme was determined. The results are shown in Table 1. The amino acid composition of the native enzyme agreed well with our previous data (Olson and Little, 1984) (not shown). For the enzyme treated in the light and which was cu. 82% inactivated, losses of arginine, tryptophan, tyrosine, phenylalanine and proline were recorded. Similar treatment, but in the dark, results in losses in only the arginine content.
- 0.6
- 0.4
Table 1. Loss of amino acids during reaction with 2,3-butancdionc in light or protected from light Amino acid
Loss in light. treated MPO
Loss in dark*
- 0.2
Arginine Tryptophan Tyrosine Phenylalanine Proline
22 33 28 13 13
26 0 0 0 0
Acllvlty
ol
0
.
I
30
.
I
60
.
I
00
I
120
I
150
.
I
lo
180
Time (mln)
Fig. 4. Influence on light on the inactivation of myeloperoxidase by 2,3-butane&one. Myeloperoxidase (1 .Omg/ml) in 50 mM sodium borate buffer, pH 8.0, was reacted with 250 mM 2,3-butanedione. Identical reaction mixtures were treated in the light (a), intensity 4001x or protected from light (I), intensity
trested MPO
‘Per cent of untreated sample. No major changes were observed in any other amino acids. Myeloperoxidase (I .Omg/ml) was incubated with 250mM 2,3-butanedione in 50mM sodium borate buffer, pH 8.0, at room temperature in light (400 lx) and protected from light (-z 5 Lx) for 180 min. Sample without reagent served as control.
NIU FEEDER WILL-EN
758 Table 2. Effect of 2,3-butanedione proteins
Protein Myeloperoxidase Lactoperoxidase Horseradish peroxidase Cytochrome c Myoglobin
on different haemo-
Per cent reduction at 280 nm
Per cent reduction at Soret peak
1.3 16.0 13.9 15.6 14.3
59.0 29.1 54.4 42.6 23.2
The proteins (myeloperoxidase, 0.25 mg/ml; lactoperoxidase, 0.22 mg/ml; horseradish peroxidase, 0.23 mg/ml; cytochrome c, 0.23 mg/ml; myoglobin 0.26 mg/ml) in 2 ml of 50 mM sodium borate buffer, pH 8.0 were incubated with 3 pl 2,3-butanedione (17 mM) in a quartz cuvette. The spectra were recorded immediately after incubation, while the modified protein spectra were recorded 1hr later.
Haem destruction in other haemoproteins by 2,3butanedione To examine whether the haem damage induced in myeloperoxidase by 2,3-butanedione in the presence of light was a special feature of myeloperoxidase or a general property of haemoproteins, four haemoproteins plus myeloperoxidase were each treated under similar conditions with the reagent. Before and after incubation, spectra were measured and changes in the Soret peak calculated. In all cases, evidence for haem damage was obvious. The greatest losses in Soret peak absorption occurred with myeloperoxidase, horseradish peroxidase and cytochrome c with somewhat smaller changes being recorded for lactoperoxidase and myoglobin (Table 2). DISCUSSION
Myeloperoxidase can be inactivated by arginine reagents, but the results suggest that the mechanism involves a photosensitizing role for the reagent, at least in the case of 2,3-butanedione. Although arginine modification occurs, no evidence of catalytically essential arginine residues was found. Indeed, incubation in the dark with 2,3-butanedione yielded a specific modification of arginine residues together with a ca. 10% activation of the enzyme. The dangers of photochemical side reactions when these diketone arginine reagents are used under other than oxygen-free or light-free conditions have been noted by other workers (see, for example, Fliss and Viswanatha, 1979; Makinen and Mlkinen, 1982). Tyrosine and tryptophan Seem to be the residues most affected during these reactions (Gripon and Hoofmann, 1981; Mlkinen et al., 1982), although other amino acid residues, especially histidine, can be modified (Miikinen and Mlikinen, 1982). With the present enzyme, damage to the haem group alone is probably sufficient to explain the inactivation. Furthermore, studies on three other haemoproteins provided evidence for marked damage to haem residues by the 2,3-butanedione photosensitized reaction. Although other workers have reported the diketone photosensitized inactivation of haem enzymes, viz. lactoperoxidase (Mlkinen and MHkinen, 1982), no mention has been made of the high sensitivity of the haem residues to destruction during the reaction. In light of our results, great
and CLIVE LITTLE
caution should be applied when using diketones to test for possible essential arginine residues in haem enzymes. We might also mention that attempts to probe for essential carboxyl residues in myeloperoxidase by using water-soluble carbodiimides in the presence of suitable nucleophiles were also thwarted as a result of destruction of haem residues (Willassen and Little, unpublished work). It seems possible that the sensitivity of haemoproteins to photochemical and chemical reactions is a much greater problem than is generally appreciated. CONCLUSIONS
The haem-enzyme myeloperoxidase was inactivated when incubated in the presence of visible light with several diketone arginine reagents. Kinetic analysis indicated that with 2,3-butanedione the inactivation involved a classic pseudo-first order reaction wherein no more than one molecule of reagent reacted per active centre. Enzyme inactivation was accompanied by changes both in the visible and U.V. spectrum of the enzyme when myeloperoxidase was incubated with diketones in the presence, but not in the absence of light. Incubation of reagent with enzyme in the dark caused a small activation of myeloperoxidase. Amino acid analyses showed that similar extents of arginine modification occurred in the presence and absence of normal laboratory lighting. In the dark, arginine was the only amino acid modified. It seems that this enzyme is inactivated by these arginine reagents by way of their photosensitizing properties and that enzyme inactivation arises mostly from damage to the haem group. Significantly, several other haemoproteins were also shown to display evidence of prothetic group damage when exposed to diketones in the presence of normal laboratory lighting. REFERENCES
Agner K. (1941) Verdoperoxidase A ferment isolated from leucocytes. Acta physiol. stand. 2, 140. Atkin C. L., Andersen M. R. and Eyre H. J. (1982) Abnormal neutrophil myeloperoxidase from a patient with chronic myelocytic leukemia. Archs Biochem. Biophys. 214, 284-292.
Aurebekk B. and Little C. (1977) Functional arginine in phospholipase C of Bacillus cereus. Int. J. Biochem. 8, 757-762.
Clark R. A. (1983) Extracellular effects of myeloperoxidasehydrogen-peroxide-halide system. Adv. Inflammation Res. 5, 107-146.
Ehrenberg A. and Agner K. (1958) The molecular weight of myeloperoxidase. Acta them. stand. 12, 95-100. Fliss H. M. and Viswanantha T. (1979) 2,3-butanedione as a photonsenitizing agent: application to a-amino acids and a-chymotrypsin. Can. J. Biochem. 57, 1267-1272. Gripon J. C. and Hofmann T. (1981) Inactivation of aspartyl proteinase by butane-2,3-dione. Biochem. J. 193, 5%5.
Harrison J. E., Pablan S. and Schultz J. (1977) The subunit structure of crystalline canine myeloperoxidase. Biochim. biophys. Acta 493, 247-259.
Klebanoff S. J. (1975) Antimicrobial mechanisms in neutrophilic polymorphonuclear leucocytes. Semin. Hematol. 12, 117-142. Levy H. M., Leber D. and Ryan E. M. (1963) Inacti-
vation of myosin by 2,4-dinitrophenol and protection by
Inactivation of myeloperoxidase adenosine triphosphate and other phosphate compounds. J. biol. Chem. 238, 36543659.
R. L. and Noyes C. M. (1984) In Chemical Protein Modification, Vol. II, pp. l-45. CRC Press, Boca Raton, Fla. Miikinen K. K. and Miikinen P. L. (1982) Photochemical inactivation of lactoperoxidase sensitized by carbonyl compounds. Eur. J. Biochem. 123, 171-178. MBkinen K. K., Mlkinen P. L., Wilkes S. H., Bayliss M. and Prescott J. M. (1982) Photochemical inactivation of aeromonas aminopeptidase by 2,3-butanedione. J. biol. Chem. 257, 17661772. Nauseef W. M., Root R. K. and Malech H. L. (1983) Biochemical and immunologic analysis of hereditary myeloperoxidase deficiency. J. clin. Invest. 71,
Lundblad
Reagents for
1297-1307.
Olsen R. L. and Little C. (1983) Purification and some properties of myeloperoxidase and eosinophil peroxidase from human blood. Eiochem. J. 209, 781-787.
159
Olsen R. L. and Little C. (1984) Studies on the subunits of human myeloperoxidase. Biochem. J. 222, 701-799. Poulos T. L. and Kraut J. (198Oa) The sterochemistry of peroxidase catalysis. J. biol. Chem. 225, 8199-8205. Poulos T. L. and Kraut J. (1980b) A hypothetical model of the cytochrome c peroxidase-cytochrome electron transfer complex. J. biol. Chem. 225, 10,322-10,330. Schultz J. and Kaminiker K. (1962) Myeloperoxidase of the leukocytes of normal human blood-I. Content and localization. Archs Eiochem. Biophys. 96, 465-467. Shindler J. S., Childs R. E. and Bardsley W. G. (1976) Peroxidase from human cervical mucus. The isolation and characterization. Eur. J. Biochem. 65, 325-331. Spade T. F. and Witkop B. (1967) Determination of tryptophan content of proteins with N-bromosuccinimide. Meth. Enzym. 11, 498-506.
Sutton B. J., Little C., Olsen R. L. and Willassen N. P. (1988) Preliminary crystallographic analysis of human myeloperoxidase. J. molec. Biol. 199, 395-396.
0020-71 IX/S9 $3.00 + 0.00 Copyright 0 1989 Pcrgamon Press plc
EFFECT OF 2,3-B~ANEDIONE ON HUMAN MYELOPEROXIDASE Nus PRDERWILLASSEN and CLIVE LITTLE Institute of Medical Biology, University of Tromse, P.O. Box. 977, N-!X#l Tromsr& Norway [Tel (083) 86560] (Received 15 November 1988)
Abstract-1. Myeloperoxidase is inhibited by various diketones that are recognized arginine reagents. 2. Although arginine residues in the enzyme were modified in both the light and the dark, enzyme inactivation occurred oniy in the presence of light. 3. Under conditions where diketones caused inactivation of myelo~ro~~, spectral studies indicated
marked damage to the haem residues of the enxyme. 4. It was concluded that diketones serve simply as photosensitizers of visible light-induced inactivation of myeloperoxidase. 5. Studies on other haemoproteins indicated the great ease with which the presence of diketones sensitized haem residues for photodestrnction.
Methods
IN’IXODUCHON
isolation and assay of enzyme
Myeloperoxidase constitutes 2-5% of dry wt of neutrophils (Schultz and Kaminker, 1962; Nauseef er of., 1983) and appears to have anti~~obi~dal functions (Klebanoff, 1975; Clark, 1983). The enxyme has a molecular weight around 140,000 (Ehrenberg and Agner, 1958) and contains two green haem prosthetic groups (Agner, 1958). The normal human enzyme seems to be a tetramer comprising two identical large (M~57,~) and two identical small (M, 10,500) subunits (Olsen and Little, 1984). The haem residues, which seem associated with the large subunit (Harrison et al., 1977; Atkin et al., 1982) are essential for the catalytic function. Little, however, is known regarding the possible participation of apoprotein in substrate binding and catalytic activity. Prompted by the control role of arginine in the proposed m~hanism of action of cytochrome c peroxidase (Poulos and Kraut, 1980a,b), we decided to investigate the effect of certain diketones on myeloperoxidase. Such reagents are commonly used for probing for “essential” arginine residues in proteins [see Aurebekk and Little (1977) and the review by Lundblad and Noyes (1984)].
Myeloperoxidase was purified from butTy coats of outdated human blood as described by Olsen and Little (1983). Myeloperoxidase with a A,r,r,fAzsor_ ratio (Rz value) between 0.81 and 0.83 was used. Enzyme of this grade of purity can be crystallii (Sutton e; a/., 1988). -Enzyme activity measurements were based on the method of Shindler et al. (1976). Assays were carried out in a 50mM sodium acetate buffer, pH 4.4, with fmal concentrations of hydrogen peroxide and 2,~-~n~i-(3~hyl~~oline~s~phonic acid) of 0.05 and I mM, respectively. The reaction was followed continuously at 412nm in a Hitachi 557 double-beam recording spectrophotometer at 22-24°C. Chemical modification reactions inactivation by l,.kyclohexanedione. Treatment of myeloperoxidase with 1,2-cyclohexanedionewas carried out at 37°C in a 2 ml reaction mixture containing myeloperoxidase (0.25 mg/ml) and 1~rnM 1,2~yclohexan~ione in
MATERIALSANDMETHODS Materials
2,2’-Azinobis(3-ethyIbenzothiazoline-6-sulfphonic acid) was purchased from Boehringer Mannheim GmbH (F.R.G.). N-Bromos~~mide was obtained from J. T. Baker (Deventer, The Ne~erlands). ~io~ycoltic acid, 2,3butanedione (freshly distilled before use), 1,2-cyclohexanedione (recrystallized) and phenylgfyoxal were from Ruka AG (Switzerland). Lactoneroxidase, horseradish neroxidase and ‘cytochrome c were from Sigma (St. Louis, MO. U.S.A.). H,O, was obtained from the local chemist as a 30% (w/v) SbluGon. All other chemicals were of analytical grade purity.
50 mM sodium borate buffer, pH 8.0. During the course of reaction aliquots were removed, diluted with the incubation buffer at room temperature (22-24”C), and assayed for remaining enxyme activity. Myeloperoxidase incubated with buffer alone served as control. Zrzactivation &y ~ny~giyoxa~. A 0.5ml solution of myelo~ro~d~ (0.5 m&ml) in 100 mM sodium bicarbonate buffer, pH 9.0 was treated by adding a 0.5 ml solution containing different concentrations of phenylglyoxal in the same buffer. Myeloperoxidase incubated with butfer alone served as a control. Aliquots were removed from both the sample and control, diluted with the incubation buffer and immediately assayed for enzyme activity. All reactions were carried out at room temperature. I~t~va~ion by .2,3-butanedione. Aliquots from a pHadjusted 2,3-butane&one-borate solution were added to myeloperoxidase (0.25- 1.Omg/ml) in 50 mM sodium borate buffer, pH 8.0 to initiate the modification reaction. This procedure was followed unless otherwise stated. Enzyme incubated with buffer alone served as untreated control. To stop the reaction, samples (IO-25~1) were withdrawn from the reaction mixture at different time periods and
755
756
NIB
PEDER
WILLASSEN and CLIVELITTLE
diluted with the incubation buffer. The enzyme activity was measured immediately afterwards. The modification was performed at room temperature. Ihmination
of samples during inactivation
Unless otherwise stated, incubation were performed in a room illuminated only by normal fluorescent light tubes. The light intensity on the reaction mixtures was measured using a HIOKI Lux Hi Tester 342 1. Amino acid analysis
Protein samples were precipitated with 20% (v/v) trichloroacetic acid (TCA), cooled and centrifuged. The precipitated proteins were washed with 6 M HCl to remove excess modification reagent. The proteins were hydrolysed for 24 hr in 6 M HCl containing 0.05% (v/v) thioglycollic acid at 110°C in glass tubes sealed under nitrogen flushing. After hydrolysis the sample were dried in a vacuum oven and dissolved directly in a 0.2 M sodium citrate buffer, pH 2.2, containing 0.5% (v/v) thiodiglycol. The quantitative amino acid analysis was performed in a JEOL JLC-6 AH amino acid analyser. Tryptophan was determined by using N-bromosuccinimide, essentially as described by Spade and Witkop (1967), on myeloperoxidase dissolved in 6 M guanidinium chloride. RESULTS
Inactivation by arginine reagents Myeloperoxidase was exposed to three different standard arginine reagents, viz. phenylglyoxal, 1,2cyclohexanedione and 2,3-butanedione. All three were able to inactivate the enzyme. Under the reaction conditions used, 2,3-butanedione inactivated the enzyme in a classic pseudo-first order kinetic manner, whereas the kinetics of inactivation by the other two reagents were more complex (Fig. 1). The inactivation by 2,3-butanedione was investigated further. In cases of pseudo-first order inactivation, the slope of plots of log [pseudo-first order rate constant] vs
10’ 0
o . n 10 20
n
c
40
30
’
SO
,.4
1.6
,.3
2.0
2.2
Time (mln)
log [reagent concentration] indicate the number of molecules of reagent reacting to inactivate one active centre in the enzyme (Levy et al., 1963). Applying such an analysis to the data for the inactivation of myeloperoxidase by 2,3-butanedione yielded a straight line of 0.72 (Fig. 2), thereby indicating that no more than one molecule of reagent was needed to inactivate each active centre in the enzyme. Nature of the chemical modiJication causing enzyme inactivation Myeloperoxidase is characterized by possessing a strong green colour. During incubation with an inactivation by 2,3-butanedione a visible bleaching of the enzyme occurred. This effect was studied more closely. Spectral studies carried out every 30min
2.4
Log(I2,3-butmadbn.]rlOOO)
IO 0
I
I
I
10
20
30
Time
Fig. 2. Inactivation of 22-24°C by treatment was incubated in light (O), 100 mM (m) and
’
Fig. 1. The rate of inactivation of myeloperoxidase with different arginine modifying reagents. Myeloperoxidase (0.25 mg/ml) was incubated in visible light (400 lx) with 100 mM 1,2_cyclohexanedione (A), 50 mM phenylglyoxal (a) and IOOmM 2,3-butanedione (0) as described in the “Materials and Methods” section.
100
1.2
’
60
40
I
I
50
60
(min)
myeloperoxidase by 2,3-butane&one. The enzyme (0.25 mg/ml) was inactivated at with 2,3-butanedione as described in “Materials and Methods”. Myeloperoxidase (400 lx) with the following concentrations of 2,3-butanedione; 25 mM (A), 50 mM 200 mM (0) in 50 mM sodium borate buffer, pH 8.0. Inset: plot of log (pseudo-first order inactivation) vs log (reagent concentration).
151
Inactivation of myeloperoxidase 0.6
I.16
0.6
I.10 0) ::
8
zi
p”
,o
9
f
5
0.4
Q
0.2
0””
Z””
4””
Wavelength
El””
0””
(nm)
Fig. 3. Effect of 2,3-butanedione on U.V. and visible spectra of myeloperoxidase. The enzyme (2 ml, OJmg/ml) in a quarts cuvette was incubated with 3~1 2,3-butanedione (17mM) and irradiated with visible light (260 lx). The spectra were measured, every 30 min for 8 hr. The ordinate on the left is from 200 to 500 nm, and the right from 500 to 640 nm. The absorption values decreased progressively during the experiment. during the reaction showed a marked diminution in the absorbance bands characteristic of the haem residue and a smaller effect on the tyrosine/ tryptophan absorbance around 280 mn was also observed (Fig. 3). Both the inactivation and the spectral changes were absent if the sample was protected from light. The changes in enzymatic activity and Rz value &,,/A& during the incubation with 2,3-butanedione in light or protected from light are shown Fig. 4. In the dark, a small, but completely reproducible activation (cu. 10%) of the enzyme
- 1.0 - 0.8
occurred. From an extrapolation of a replot of the data in the form of percentage inactivation against percentage loss of absorbance at 430 or 280 nm (not shown), it was estimated that the total inactivation (in light) corresponded to a 50% decrease in the absorbance due to the haem residues and about 20% at 280nm. In order to test for the modification of specific amino acid residues, the amino acid composition of native and 2,3-butanedione-treated (in the light and dark) enzyme was determined. The results are shown in Table 1. The amino acid composition of the native enzyme agreed well with our previous data (Olson and Little, 1984) (not shown). For the enzyme treated in the light and which was cu. 82% inactivated, losses of arginine, tryptophan, tyrosine, phenylalanine and proline were recorded. Similar treatment, but in the dark, results in losses in only the arginine content.
- 0.6
- 0.4
Table 1. Loss of amino acids during reaction with 2,3-butancdionc in light or protected from light Amino acid
Loss in light. treated MPO
Loss in dark*
- 0.2
Arginine Tryptophan Tyrosine Phenylalanine Proline
22 33 28 13 13
26 0 0 0 0
Acllvlty
ol
0
.
I
30
.
I
60
.
I
00
I
120
I
150
.
I
lo
180
Time (mln)
Fig. 4. Influence on light on the inactivation of myeloperoxidase by 2,3-butane&one. Myeloperoxidase (1 .Omg/ml) in 50 mM sodium borate buffer, pH 8.0, was reacted with 250 mM 2,3-butanedione. Identical reaction mixtures were treated in the light (a), intensity 4001x or protected from light (I), intensity
trested MPO
‘Per cent of untreated sample. No major changes were observed in any other amino acids. Myeloperoxidase (I .Omg/ml) was incubated with 250mM 2,3-butanedione in 50mM sodium borate buffer, pH 8.0, at room temperature in light (400 lx) and protected from light (-z 5 Lx) for 180 min. Sample without reagent served as control.
NIU FEEDER WILL-EN
758 Table 2. Effect of 2,3-butanedione proteins
Protein Myeloperoxidase Lactoperoxidase Horseradish peroxidase Cytochrome c Myoglobin
on different haemo-
Per cent reduction at 280 nm
Per cent reduction at Soret peak
1.3 16.0 13.9 15.6 14.3
59.0 29.1 54.4 42.6 23.2
The proteins (myeloperoxidase, 0.25 mg/ml; lactoperoxidase, 0.22 mg/ml; horseradish peroxidase, 0.23 mg/ml; cytochrome c, 0.23 mg/ml; myoglobin 0.26 mg/ml) in 2 ml of 50 mM sodium borate buffer, pH 8.0 were incubated with 3 pl 2,3-butanedione (17 mM) in a quartz cuvette. The spectra were recorded immediately after incubation, while the modified protein spectra were recorded 1hr later.
Haem destruction in other haemoproteins by 2,3butanedione To examine whether the haem damage induced in myeloperoxidase by 2,3-butanedione in the presence of light was a special feature of myeloperoxidase or a general property of haemoproteins, four haemoproteins plus myeloperoxidase were each treated under similar conditions with the reagent. Before and after incubation, spectra were measured and changes in the Soret peak calculated. In all cases, evidence for haem damage was obvious. The greatest losses in Soret peak absorption occurred with myeloperoxidase, horseradish peroxidase and cytochrome c with somewhat smaller changes being recorded for lactoperoxidase and myoglobin (Table 2). DISCUSSION
Myeloperoxidase can be inactivated by arginine reagents, but the results suggest that the mechanism involves a photosensitizing role for the reagent, at least in the case of 2,3-butanedione. Although arginine modification occurs, no evidence of catalytically essential arginine residues was found. Indeed, incubation in the dark with 2,3-butanedione yielded a specific modification of arginine residues together with a ca. 10% activation of the enzyme. The dangers of photochemical side reactions when these diketone arginine reagents are used under other than oxygen-free or light-free conditions have been noted by other workers (see, for example, Fliss and Viswanatha, 1979; Makinen and Mlkinen, 1982). Tyrosine and tryptophan Seem to be the residues most affected during these reactions (Gripon and Hoofmann, 1981; Mlkinen et al., 1982), although other amino acid residues, especially histidine, can be modified (Miikinen and Mlikinen, 1982). With the present enzyme, damage to the haem group alone is probably sufficient to explain the inactivation. Furthermore, studies on three other haemoproteins provided evidence for marked damage to haem residues by the 2,3-butanedione photosensitized reaction. Although other workers have reported the diketone photosensitized inactivation of haem enzymes, viz. lactoperoxidase (Mlkinen and MHkinen, 1982), no mention has been made of the high sensitivity of the haem residues to destruction during the reaction. In light of our results, great
and CLIVE LITTLE
caution should be applied when using diketones to test for possible essential arginine residues in haem enzymes. We might also mention that attempts to probe for essential carboxyl residues in myeloperoxidase by using water-soluble carbodiimides in the presence of suitable nucleophiles were also thwarted as a result of destruction of haem residues (Willassen and Little, unpublished work). It seems possible that the sensitivity of haemoproteins to photochemical and chemical reactions is a much greater problem than is generally appreciated. CONCLUSIONS
The haem-enzyme myeloperoxidase was inactivated when incubated in the presence of visible light with several diketone arginine reagents. Kinetic analysis indicated that with 2,3-butanedione the inactivation involved a classic pseudo-first order reaction wherein no more than one molecule of reagent reacted per active centre. Enzyme inactivation was accompanied by changes both in the visible and U.V. spectrum of the enzyme when myeloperoxidase was incubated with diketones in the presence, but not in the absence of light. Incubation of reagent with enzyme in the dark caused a small activation of myeloperoxidase. Amino acid analyses showed that similar extents of arginine modification occurred in the presence and absence of normal laboratory lighting. In the dark, arginine was the only amino acid modified. It seems that this enzyme is inactivated by these arginine reagents by way of their photosensitizing properties and that enzyme inactivation arises mostly from damage to the haem group. Significantly, several other haemoproteins were also shown to display evidence of prothetic group damage when exposed to diketones in the presence of normal laboratory lighting. REFERENCES
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Lundblad
Reagents for
1297-1307.
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159
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Sutton B. J., Little C., Olsen R. L. and Willassen N. P. (1988) Preliminary crystallographic analysis of human myeloperoxidase. J. molec. Biol. 199, 395-396.