SPECTROSCOPIC INVESTIGATIONS ON THE HIGHLY PURIFIED LACTOPEROXIDASE FE(III)-HEME CATALYTIC SITE

Purification of the lactoperoxidase (LPO) major cationic isoenzyme was significantly improved by the use of preparative chromatographic and electrophoretic methods combined with analytical electrophoretic techn iques and image processing. A detailed report is given of the experime ntal procedure. Furthermore, electron paramagnetic resonance has playe d a fundamental role in evaluating the enzyme purity against lactoferr in and minor LPO isoenzyme components in setting the final steps of th e purification. With the aim to completely clarify the Fe(III)-heme hi gh-spin nature of the native LPO, two samples of lactoperoxidase, LPO1 and LPO2 (RZ = 0.95) from farm and commercial milk, respectively, wer e purified and characterized in particular by electron paramagnetic re sonance (EPR) spectroscopy, in comparison with a commercial preparatio n (LPOs). The LPO1 EPR spectrum, at physiological pH, is clearly indic ative of the presence of an iron(III)-heme high-spin catalytic site in the native enzyme. On the contrary, in the LPO2 spectrum a thermal eq uilibrium between high- and low-spin iron(III)-heme species is present . The low-spin component of the spectrum has been assigned to an LPO-N O, adduct due to the presence of some nitrite impurities originating e ither from commercial unpasteurized milk or from external sources. The LPOs EPR spectrum shows the presence of some spurious lines in the g congruent to 6 and 4 regions due to the minor LPO isoenzyme components and to lactoferrin, respectively. The LPO EPR spectra previously repo rted in the literature contain a variable number of spurious lines in the g congruent to 4 and 2 regions as a consequence of lactoferrin imp urity and LPO low-spin adducts with endogenous or exogenous anions. Fu rthermore, the interaction of LPO with its native substrate (the thioc yanate anion), which previously was shown by NMR and EPR (at high subs trate concentration) spectroscopies, has been confirmed by EPR at low temperature and low substrate concentration and by optical spectroscop y at room temperature and high substrate concentration as a function o f pH. The LPO activity at optimum pH (congruent to 4-5) has been measu red in phosphate and acetate buffer using as an oxidizable substrate t he system dimethylamino benzoic acid 3-methyl-2-benzothiazolinone hydr azone hydrochloride monohydrate (DMAB-MBTH), which was considered a go od chromogen for other peroxidases such as HRP and zucchini peroxidase s. The LPO vs. SCN- activity at optimum pH (congruent to 5.5) has been measured in phosphate and acetate buffer. Kinetic studies in the pres ence of the DMAB-MBTH chromogen show a noncompetitive inhibition betwe en hydrogen peroxide and thiocyanate, in particular at optimum SCN- su bstrate activity around pH 5.5. Spectroscopic results together with ac tivity and kinetic data seem to indicate the insertion of thiocyanate as a ligand bridge between Fe(III)-heme and N3H imidazole of the dista l histidine to give a small Fe(III) interaction at room temperature, w hich became stronger at low temperature. Previous NMR and preliminary EPR measurements suggested the heme pocket site close to the Fe(III) i on as the SCN- binding site.