Conformational and thermal stability of mature dimeric human myeloperoxidase and a recombinant monomeric form from CHO cells (1)
Myeloperoxidase is an enzyme that prefers small anionic molecules as electron donors, such as halides (chloride, bromide, iodide), thiocyanate and nitrite. Myeloperoxidase is secreted at inflammatory sites from stimulated polymorphonuclear leukocytes and also monocytes. Myeloperoxidase is the only vertebrate peroxidase that is a (symmetric) homodimer with the two large polypeptides being linked by a disulfide bond.
Upon incubation of both proteins with guanidinium hydrochloride (GdnHCl) and monitoring the spectral features of the metalloprotein in the visible region, the stability of the heme cavity was probed. GdnHCl was used because urea was less efficient in MPO unfolding. Even at highest GdnHCl concentration at least part of the MPO-typical heme to protein bonds remain intact and enable (unspecific) heme coordination with the protein.
Unfolding of MPO by urea was clearly incomplete. As a consequence only GdnHCl-mediated unfolding in the absence and presence of DTT was analyzed. MPO unfolding by GdnHCl was very slow and even in 6 M GdnHCl the transition took more than 15 min. Incubation of MPO to GdnHCl at the determined Cm concentrations completely abolished the peroxidase and chlorination activity within minutes. However, dilution to 0.25 M GdnHCl could slowly restore partially the enzyme activity within ~2 h depending on the actual incubation time with GdnHCl. Very similar effects were observed with MPO and proMPO and with both peroxidase and chlorination activity (15 min exposure: 50–60%; 18 h exposure: 10–15% recovery).
Upon continuous increase of temperature (10 to 90 °C) the Soret maximum of leucocyte MPO obtained complete breakage of all three heme to protein linkages, which was not observed in unfolding experiments with GdnHCl even in the presence of DTT. In temperature-mediated unfolding experiments all samples contained 0.5 M GdnHCl in order to avoid temperature-induced precipitation of both proteins. At this denaturant concentration neither MPO nor proMPO showed structural (and spectral) modifications compared to the native proteins.
Melting of α-helices of recombinant proMPO followed also a two- state transition with a Tm of 70.5 °C which corresponds to that observed in monitoring the unfolding of the heme cavity (Table 2). Analysis of thermal unfolding of proMPO under reducing conditions did not give reliable data due to protein aggregation even in the presence of 0.5 M GdnHCl.
After incubation at 80 °C for 15 min leukocyte MPO had still about 50% residual activity, whereas proMPO more or less lost its catalytic functions.
Deconvolution of data might suggest a non-two-state unfolding and the presence of two distinct domains with close Tm values (MPO: 85.3 and 88.5°C; recombinant proMPO: 74.3 and 77.6 °C) and similar thermodynamic parameters.
Under reducing conditions MPO is unstable and easily prone to inactivation. The disulfide bridges significantly contribute to the structural integrity of the light and heavy chain within a protomer, and one cystine additionally covalently links the two monomers in leukocyte MPO. Comparison study of biological functionality of glycosylated and deglycosylated recombinant proMPO showed that glycosylation is required for optimal enzymatic activity.
Leukocyte MPO is an extremely stable heme peroxidase with calculated Tm and Cm values of >80 °C and >4.3 M GdnHCl.