Purification, Properties, and Amino Acid Sequence of a Hemoglobinuria-Inducing Phospholipase A2, MiPLA-1, from Micropechis ikaheka Venom

Abstract

Dark-colored urine is one of the clinical symptoms of envenomation by Micropechis ikaheka (New Guinea small-eyed snake). We have purified a phospholipase A2, MiPLA-1, which induces dark-colored urine in experimental mice, to homogeneity. The analysis of the dark-colored urine by electrophoresis and N-terminal sequence determination indicated that the color of mouse urine is due to hemoglobin in the urine but not myoglobin. MiPLA-1 is the first hemoglobinuria-inducing toxin. Insignificant hemolytic activity of MiPLA-1 indicates that hemoglobinuria is not due to lysis of erythrocytes by MiPLA-1. This suggests that hemoglobinuria induced by MiPLA-1 may be due to kidney leakage caused by unknown mechanisms. MiPLA-1 also showed other biological effects, including myotoxicity as well as anticoagulant and antiplatelet effects. Structural studies show that MiPLA-1 is a basic protein with a molecular mass of 14041.60 ± 1.78 as determined by electrospray mass spectrometry. We have determined the complete amino acid sequence of MiPLA-1. It is a 124-amino-acid protein with a “pancreatic loop” and belongs to group IB phospholipase A2 enzymes. Two short segments flanked by proline brackets are found in the sequence of MiPLA-1. These segments are on the surface of the molecule and hence may be involved in protein–protein recognition.