Spectroscopic investigations on the highly purified lactoperoxidase Fe(III)-heme catalytic site

Abstract

Purification of the lactoperoxidase (LPO) major cationic isoenzyme was significantly improved by the use of preparative chromatographic and electrophoretic methods combined with analytical electrophoretic techniques and image processing. A detailed report is given of the experimental procedure. Furthermore, electron paramagnetic resonance has played a fundamental role in evaluating the enzyme purity against lactoferrin and minor LPO isoenzyme components in setting the final steps of the purification. With the aim to completely clarify the Fe(III)-heme high-spin nature of the native LPO, two samples of lactoperoxidase, LPO1 and LPO2 ( RZ = 0.95) from farm and commercial milk, respectively, were purified and characterized in particular by electron paramagnetic resonance (EPR) spectroscopy, in comparison with a commercial preparation (LPOs). The LPO1 EPR spectrum, at physiological pH, is clearly indicative 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 equilibrium between high- and low-spin iron(III)-heme species is present. The low-spin component of the spectrum has been assigned to an LPO-NO − 2 adduct due to the presence of some nitrite impurities originating either from commercial unpasteurized milk or from external sources. The LPOs EPR spectrum shows the presence of some spurious lines in the g ≊ 6 and 4 regions due to the minor LPO isoenzyme components and to lactoferrin, respectively. The LPO EPR spectra previously reported in the literature contain a variable number of spurious lines in the g ≊ 4 and 2 regions as a consequence of lactoferrin impurity and LPO low-spin adducts with endogenous or exogenous anions. Furthermore, the interaction of LPO with its native substrate (the thiocyanate anion), which previously was shown by NMR and EPR (at high substrate concentration) spectroscopies, has been confirmed by EPR at low temperature and low substrate concentration and by optical spectroscopy at room temperature and high substrate concentration as a function of pH. The LPO activity at optimum pH ( ≊ 4–5) has been measured in phosphate and acetate buffer using as an oxidizable substrate the system dimethylamino benzoic acid 3-methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (DMAB-MBTH), which was considered a good chromogen for other peroxidases such as HRP and zucchini peroxidases. The LPO vs. SCN − activity at optimum pH ( ≊5.5) has been measured in phosphate and acetate buffer. Kinetic studies in the presence of the DMAB-MBTH chromogen show a noncompetitive inhibition between hydrogen peroxide and thiocyanate, in particular at optimum SCN − substrate activity around pH 5.5. Spectroscopic results together with activity and kinetic data seem to indicate the insertion of thiocyanate as a ligand bridge between Fe(III)-heme and N 3H imidazole of the distal histidine to give a small Fe(III) interaction at room temperature, which became stronger at low temperature. Previous NMR and preliminary EPR measurements suggested the heme pocket site close to the Fe(III) ion as the SCN − binding site.