Immunoaffinity purification of human thromboxane synthase.

A recently produced monoclonal antibody against human thromboxane synthase was used to purify the enzyme from platelets in a one-step procedure with good yields. The isolated protein exhibited a single band of about 58 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained one heme/mol. Although the visible spectrum of the oxidized enzyme displayed a peak at 418 nm like the previously isolated enzyme after dithionite reduction and CO addition, it shifted to 419 nm but not to 450 nm where only a small shoulder could be detected. Its catalytic activity was only 1-5% of the previous preparations, but with the same Km of about 10 microM and a ratio of thromboxane B2: 12-hydroxyheptadecatrienoic acid of 1:1. Studies with EPR spectrometry and inhibitors confirmed that only a minor part of the enzyme was in its native heme-thiolate conformation, whereas the major part had been converted to the inactive P420 form by the elution procedure. The amino acid analysis revealed 46% hydrophobic residues. According to the sequence of 26 amino acids from the N-terminus and two tryptic peptides no homology to one of the cytochrome P450 monooxygenases, or to cyclooxygenase, or to prostacyclin synthase was detected.