108s BiochemicalSocietyTransactions (1 992) 20

EPR differences between human myeloperoxidase isoenzymes.

Table 1.

CHRIS E. COOPER* AND EDDIE ODELLx

Myeloperoxidase complexes formed as in Figure 1.

*Division of Biomolecular Sciences, King's College London, Campden Hill Road, London W8 7AH, UK # Dept. Oral Medicine and Pathology, UMDS, Guy's Hospital, London Bridge, London SEI 9RT

lsoenzyme

gx

gy

&

Chloride comdex I II 111

6.82 6.85 6.84

5.035 5.01 5.01

I .953 1.95 I .95

Nitrite ComDlex I I1 111

2.563 2.547 2.545

2.304 2.308 2.309

1.812 1.816 1.816

Myeloperoxidase is the most abundant haem protein in neutrophils, amounting to 2-5%of total cell protein. It plays a vital role in their anti-bacterial function 11 I. The holoenzyme contains two heavy and two light polypeptide subunits and two spectrally indistinguishable chlorin-type haems 121. When purified the enzyme exists in a femc high spin form with characteristic electron paramagnetic resonance (EPR) g-values at gA.8, 5.0 and 1.95. As is the case with lactoperoxidase (31,the addition of nitrite to the enzyme converts the haem to a low spin femc nitrite complex with new g-values of 2.55, 2.3 and 1.8, even in the presence of relatively high levels of the reducing agent sodium dithionite (Figure I , see also 141).A small proportion of a ferrous-nitrosyl complex is seen at g=2,but to convert the enzyme completely into this form presumably requires a higher ratio of dithionitehitrite, as with lactoperoxidase (3.41. Myeloperoxidase can be purified by cation exchange chromatography into three major isoenzyme fractions (51, due to primary sequence variation, 161 and consequent differences in glycosylation 171. Three distinct cDNA clones have been isolated for the enzyme, derived from alternative splicing of the mRNA transcript from a single gene 181.

lsoenzyme I differs from isoenzymes II and 111 in substrate reactivity 191. It has been reported that the EPR and pyridine heaemochrome spectra are identical for all three isoenzymes (91. However, Table I shows that isoenzyme I shows a smaller rhombic distortion than the other isoenzymes in the high spin chloride complex. Moreover it shows a larger rhombic distortion in the low spin nitrite complex. The same small difference in the chloride complex was reported by Wright et al. 191, but not considered significant. However, our confirmation of this observation and the additional difference in the low spin nitrite spectra reveals a significant difference in the EPR spectra of isoenzyme 1. In agreement with Wright et al. 191. however, we see no difference in the position of the optical spectra of the various isoenzyme ligand complexes. It is interesting that the isoenzyme with different reactivity is also that with distinct EPR spectra. The differences between the spectra of isoenzyme I and isoenzymes llllll are too small to be due to a change in the haem ligand at the active site. They are comparable with the minor changes induced by a change in the environment of the enzyme (e.g. due to changes in pH, ionic strength, buffer solute). These EPR spectra are therefore reporting on subtle differences in the environment of the haem group. between the isoenzyrnes. CEC is grateful for a King's College Research Fellowship.

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Fig. 1

(GAUSS1

EPR spectra of chloride and nitrite comolexes of Durified mveloDeroxidase

9SpM myeloperoxidase in lOmM Na-Hepes, pH 7.0 + IOOmM NaCl (chloride complex) or + 5mM sodium dithionitdl0mM sodium nitrite (nitrite complex). W K ('onditions: Temperature 30K. Microwave Frequency = 9.36 GHz, microwave power 2OmW. modulation frequency 100KHz. modulation amplitude IOG. receiver gain 1.6 x 105. time constant 0.33% swccp time 24 Gauss/s. Signals are average of 3 scans.

EPR uarameters for mveloDeroxidase iscenzvme S

I

1. Klebanoff, S.J. & Hamon, C.B. (1972) J. Reticuloendothel. SOC. 12, 170-1%. 2. Ikeda-Saito, M. & Prince, R.C. (1985) J. Biol. Chem. 260, 830 1-8305. 3. Lukat, G.S., Rodgers, K.R. & Goff, H.M. (1987) Biochemistry 26, 6927-6932. 4. Bolscher, B.G.J.M. & Wever, R. (1984) Biochirn. Biophys. Acta 791, 75-81. 5 . Miyasaki. K.T., Wilson, M.E., Cohen. E., Jones, P.C. & Genco, R.J. (1986) Arch. Biochem. Biophys. 246, 751764. 6. Pember, S.O., Shapira, R. & Kinkade Jr., J.M.K. (1983) Arch. Biochem. Biophys. 221, 391-403. 7. Selsted. M.E. & Novotny, M.J. (1988) Blood 72, 152a. 8. Hashinaka, K., Nishio,C., Hur, S.-J., Sakiyama, F. Tsunasawa. S. & Yamada. M. (1988) Biochemistrv 27, 5906-5914.' 9. Wright. J., Bastian, N., Davis, T.A., Zuo, C., Yoshimoto, S., Orme-Johnson, W.H. & Tauber, A.I. (1990) Blood 75,

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