Isolation and Biochemical Characterization of Transferring From Tsetse Fly, Glossina morsitan centralis

Abstract

Tsetse flies are biting insects that transmit trypanosomiasis to man and domesticated animals. Trypanosomiasis is commonly known as Nagana in cattle, Surra in camels and Sleeping sickness in humans. No sustainable control methods for this disease are currently available and yet in tropical Africa it is one of the most important vector- borne disease of medical and veterinary importance. i Tsetse flies ingest large amounts of blood from their hosts and therefore face an oxidative challenge due to iron release. Iron binding proteins are known to play a major role in controlling this toxicity by regulating iron upta.ke, transport and storage. Understanding how this detoxification mechanisms work_ can be very important in designing novel control strategies. This study focused on purification and biochemical characterization of transferrin from Glossina morsilan centralis. To achieve these goal, a number of experiments were carried out and this included screening the hemolymph and midgut samples for iron binding proteins using Ferene S (0.75mM 3-[2-Pyridyl]-5,6-bis (2-[-Fury] sulfonic acid])- l,2,4-Triazine, 2% acetic acid, 0.1% thioglycollic acid). Ferene S staining of hemolymph samples on polyacrylamide gels indicated presence of two distinct iron binding proteins, one of high molecular weight (490 kDa) and the other of low molecular weight (140 kDa). However, midgut samples did not stain significantly for iron binding proteins. The first step of transferrin purification fi'om the tsetse hemolymph involved KBr density gradient ultracentrifugation. This was followed by preparatory gel electrophoresis and electroelution of the lower molecular weight Ferene S stained iron binding protein. The electroeluted protein was applied on Concanavalin A affinity column and the bound fiaction eluted with on-methyl-D-mannopyranoside. This fraction was finally subjected to 95% ammonium sulphate precipitation. The homogeneity of the purified protein was confirmed by silver stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional (2-D) gel electrophoresis. Separation on SDS-PAGE indicated that the protein had an approximate molecular weight of 80 kDa. This indicates conservation in terms of size as it had been shown by earlier studies that 121 amino acid residues are conserved in vertebrate, cockroach and Manduca sexta transferrin. However native gels indicated a higher molecular weight of approximately 140 kDa and this difference was attributed to the structure and charge properties of the protein in the native state. The isoelectric point of transferrin was estimated at 6.5 and this is consistent with previous studies that have shown that insect hemolymph proteins have a pI of 4 - 8. Staining with,P‘eriodic acid Schifi‘ reagent and binding to con A affinity column revealed that transferrin was glycosylated and of high mannose type, respectively. Absorption spectroscopy spectrum of transferrin showed two maxima (565,370 nm) and a weak absorption at 280 nm. Key words: T ranrsferrin, Iron binding, Glossina morsitan centralis.