Hematin Detoxification Assay for Necator americanus Glutathione S Transferase -1 (Na-GST-1): assessing the heme detoxification function of nu-class glutathione-s-transferases.

Human hookworm infects more than 700 million people worldwide and Necator americanus causes approximately 85% of those infections. The hookworm ingests erythrocytes containing hemoglobin and the hemoglobin is digested to Heme and Globin by the hookworm\u27s gut enzymes. The breakdown of Globin provides essential nutrition to the hookworm. However, Heme (ferroprotoporphyrin) which contains iron is a potent producer of reactive oxygen species (ROS) and is toxic to the hookworm. It has long been hypothesized that the nu-class hookworm Glutathione S-Transferase called the Necator americanus Glutathione S-Transferase-1 (Na-GST-1) binds and detoxifies Heme, and therefore, plays a significant role in reducing the toxic reactive oxygen species (ROS) and iron burden of the hookworm. To prove this hypothesis, we have developed a unique Hematin Detoxification Assay, which measures the release of the free iron from the degraded Hematin (ferriprotoporphyrin) in presence of Na-GST-1. This assay utilizes hydrogen peroxide (H2O2), a potent oxidizer, to degrade Hematin to Biliverdin, ferric iron (Fe+3) and carbon monoxide. Ascorbic acid is then used to reduce the ferric iron (Fe+3) to ferrous iron (Fe+2). The levels of the ferrous iron (Fe+2) are then measured in the presence of ferrozine (free iron detection reagent) using a spectrophotometer (562 nm). Using this assay, a statistically significant (p = 0.0079) reduction in the iron released from 250 µM of Hematin was found in the presence of 5 µg of Na-GST-1 when compared to the iron released from 250 µM of Hematin alone. However, human placental GST did not (p = 0.578) prevent the release of free iron from 250 µM of Hematin. This in-vitro assay showed that Na-GST-1 specifically binds and protects Hematin from degradation. Based on neutralizing this detoxification function, an Alhydrogel® adjuvanted recombinant Na-GST-1 vaccine is currently under clinical development. We believe that this novel assay will also be very useful for assessing the detoxification activity of nu-class GSTs from other hematophagous helminths