Identification of the Major Arsenic-Binding Protein in Rat Plasma As the Ternary Dimethylarsinous−Hemoglobin−Haptoglobin Complex

Abstract

Chronic exposure to arsenic causes a wide range of diseases such as hyperkeratosis, cardiovascular diseases, and skin, lung, and bladder cancers, and millions of people are chronically exposed to arsenic worldwide. However, little is known about the mechanisms underlying these toxic actions. The metabolism of arsenic is essential for understanding the toxic actions. Here, we identified the major arsenic-binding protein (As-BP) in the plasma of rats after oral administration of arsenite by the use of two different HPLC columns, gel filtration and anion exchange ones, coupled with an inductively coupled argon plasma mass spectrometer (ICP MS). The molecular mass of the As-BP was estimated to be 90 kDa based on results using the former column, and arsenic bound to this protein only in the form of dimethylarsinous acid (DMA (III)) in the plasma in vivo. In addition, the purified As-BP was shown to consist of two different proteins, haptoglobin (Hp) of 37 kDa (three bands) and the hemoglobin (Hb) alpha chain of 14 kDa (single band), using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), respectively, suggesting that the As-BP was the ternary DMA (III)-Hb-Hp complex. To confirm the present observations, an arsenic-binding assay was carried out in vitro . Although DMA (III) bound directly to fresh rat plasma proteins, they were different from that identified in vivo. However, when a DMA (III)-exposed rat RBC lysate (DMA (III) binds to Hb in rat RBCs) was added to control rat plasma, a new arsenic peak increased at the expense of the arsenic-Hb one. Furthermore, this new arsenic peak was consistent with the As-BP identified in the plasma in vivo, suggesting that arsenic bound to Hb further binds to haptoglobin (Hp), forming the ternary As-Hb-Hp complex.