Identification of Novel Peroxidases In Vivo

There are many unanswered questions about the bodily effects of oxidants generated by neutrophils upon their activation during the inflammatory response. Though the primary function of such oxidants lies in the attack on foreign microorganisms, they also play a role in tissue damage associated with inflammatory diseases ranging from Alzheimer's disease to cancer.

In order to gain a better understanding of neutrophil oxidant products in the body, the mechanisms behind oxidant generation must be decoded. The heme enzyme myeloperoxidase (MPO) plays a fundamental role in oxidant production by neutrophils. MPO can act as a catalyst for the generation of hypochlorous acid, more commonly known as bleach.

Recently, it has been shown that MPO from activated neutrophils generates reactive nitrogen species (RNS) as well as hypochlorous acid. However, there is little evidence that MPO-derived RNS are being generated in vivo.

Genetic engineering offers a powerful approach to test the hypothesis that MPO generates RNS in vivo; MPO-deficient mice were recently created. Surprisingly, phagocytes isolated from these animals were still able to generate RNS. Furthermore, the reaction appeared to be peroxidase-dependent. This suggests that there is an unknown peroxidase present in MPO-deficient neutrophils that allows these pathways to continue.

The purpose of this project was to optimize an assay that characterizes peroxidase activity in protein samples with the ultimate goal of testing the peroxidase activity of wild-type and MPO-deficient neutrophils. Testing this enzyme activity would eventually lead to determining the peroxidase responsible for generating reactive nitrogen species in MPO-deficient mice.

An in-gel peroxidase activity system was developed. A unique cationic gel system (Kettle) was used in order to separate the strongly cationic peroxidases and retain enzymatic activity. In selecting an optimal stain-enhancing substrate, it was discovered that nitrite reacted with another component of the stain resulting in extremely faint bands; therefore, bromide was used as the optimal substrate for stain enhancement. In optimizing the sensitivity of the assay, it was discovered that stains containing bromide were able to reveal activity bands for 0.5 µg quantities of purified myeloperoxidase.

After creating an optimal assay, neutrophil samples from wild type and MPO-deficient mice were tested for peroxidase activity. However, because protein samples from the neutrophils were extremely dilute, results were inconclusive.

Although this highly effective peroxidase activity assay could not offer insight into the original research question, this assay was used to investigate the presence of peroxidases in the brain. Many lines of evidence implicate peroxidase activity in the pathogenesis of Alzheimer's and Parkinson's disease. Results with brain protein in the assay suggested that a peroxidase was indeed present in brain protein extract.

These preliminary results provide significant insight into future possibilities for research. In order to further explore peroxidase activity in the brain, immunohistochemistry may be performed on brain tissue to detect which cell types display peroxidase activity. In addition, various regions of the brain should be tested for peroxidase activity. Comparing diseased brains to those normally functioning would also be beneficial.

This project has been successful in creating an assay that answers only introductory questions to research topics, but it has opened the door for the investigation of many future research problems.