Large-Scale Purification of Myeloperoxidase from HL60 Promyelocytic Cells: Characterization and Comparison to Human Neutrophil Myeloperoxidase (1)
Myeloperoxidase (MPO) is a heme-containing protein found in the primary granules of neutrophils and monocytes. It is an extremely abundant protein, comprising up to 5% of the total protein in neutrophils. The MPO consists of two 54-kDa heavy chains and two 14-kDa light chains, resulting in a protein approximately 150-kDa. However, there is a hemi-myopeloxidase consisting of one heavy chain and one light chain, as shown 80-kDa. Neutrophil myeloperoxidase is extremely stable, such as the enzyme activity remaining fully after 5 min at 65°C and to lose only 10% activity after 5 min at 75°C.
Packed cell pellets of 5–13 ml (approximately (0.5– 1.5) x 10^10 total cells produced in a week) were lysed by Dounce homogenization in sodium phosphate buffer. Following centrifugation, the particulate fraction was solubilized with 1% CTAB. Buffer exchange was carried out by dialysis and the sample was loaded onto a concanavalin A column. Myeloperoxidase was eluted from the column using a buffer containing methyl a-D-mannopyranoside. Eighty percent of the total myeloperoxidase peroxidation activity was located in the CTAB solubilized cellular pellet while only 20% was located in the cytosolic fraction.
Fractions containing myeloperoxi-dase were pooled and precipitated using ammonium sulfate. The pellet was resuspended in 30 mM sodium acetate, pH 4.7 containing 100 mM sodium chloride and loaded onto a Superose 6 –Superose 12 columns. The total amount of myeloperoxidase purified per cell pellet ranged from 10 to 18 mg. This represents a 30 –50% yield intotal myeloperoxidase.
With 4-20% SDS-PAGE analysis, both the large and small subunits were detected at 60 kDa and 14 kDa respectively.
Anti-MPO monoclonal antibody recognized the large unit only, instead anti-MPO polyclonal antibodies bound to both of the large and small units.
The following are purification methods.
Purification of HL60 myeloperoxidase.
Cell pellets were resuspended in Buffer A (6.7 mM sodium phosphate(pH 6.0), 1 mM MgCl2, 3 mM NaCl, 0.5 mM PMSF) ata ratio of 10 ml of buffer to 1 ml of cell pellet. The cells were lysed by Dounce homogenization on ice and then centrifuged at 20,000gfor 30 min. The pellets were resuspended in Buffer A. CTAB was added to a final concentration of 1%, and the mixture was stirred vigorously for 2h at 4°C. The insoluble material was removed by centrifugation at 20,000gfor 20 min at 4°C. The solubilized material was dialyzed against Buffer B (100 mM sodium acetate (pH 6.3), 100 mMNaCl) for 5h at 4°C. CaCl2, MgCl2, and MnCl2 were then added to a final concentration of 1 mM each. Thematerial was mixed end-over-end with 5 ml of con-canavalin A overnight at 4°C. The resin, which at this point was dark green, was poured into a Bio-Rad Econo Column. The myeloperoxidase was eluted from the con-canavalin A with 5-ml pulses of 500 mM methyl alpha-D-mannopyranoside in Buffer B plus CaCl2, MgCl2, and MnCl2. The myeloperoxidase-containing fractions were determined both by the dark green color of the fractions and by A430 values. The fractions were then pooled and adjusted to 85% ammonium sulfate by the addition of dry ammonium sulfate. The sample was rotated end-over-end for 1–2 h at 4°C and then centrifuged at 35,000g for 30 min. The precipitated myeloperoxidase was resuspended in no more than 1 ml of Buffer C (25mM sodium acetate (pH 4.7), 100 mM NaCl) and loaded onto tandem Superose 6 and Superose 12 columns (both 50 x 2.5 cm). The myeloperoxidase was eluted in Buffer C at a flow rate of 0.5 ml/min. Dry Ammonium sulfate was added to the pooled fractions toa final concentration of 70% ammonium sulfate. Thesample was rotated end-over-end for 1h at 4°C and then centrifuged at 35,000gfor 30 min. The precipitated myeloperoxidase was resuspended in MilliQ water at 20 mg/ml, dialyzed against water to remove any remaining ammonium sulfate, and then frozen at -20°C.