Electrophoresis release of hemoglobin from living red blood cells

Abstract

Nowadays, proteomics research is almost always made by lysing cells to release protein first and then to analyze the protein solution in many ways, the lytic product of red blood cells is also called hemolysate, the main components of which are hemoglobin A 1 , A 2 , A 3 and carbonic anhydrase (CA), etc. In our laboratory, intact (unlysed) living red blood cells are used for direct electrophoresis so as to observe the released hemoglobin (Hb). Thus an electophoresis release experiment has been established with starch-agarose mixed gel. Electrophoresis release can be classified as initial release and re-release. In the former, the electropheresis is completed by switching on once when the release of Hb can be seen. In the latter, the electrophoresis is switched off and then switched on, when Hb release can be seen again. In our laboratory, HbA 2 phenomenon was observed in the initial release experiment and HbA 2 and HbA 1 interaction was seen in Hb cross eletrophoresis. In a two-way diagonal electrophoresis of the initial release type, HbA 2 and HbA 1 were found to exist in binding in the red blood cells. SDS-PAGE and mass spectrometry were conducted for the released products in the HbA 2 phenomenon of the electrophoresis and it was found that Prx-2 (peroxiredoxin-2) may be involved in the formation of HbA 2 phenomenon. In the re-release experiment, it was found that Hb mostly had “multi-band re-release phenomenon”. Based on this research, we designed a series of relevant experiments such as isotonic re-release, hypotonic re-release, full re-release from isolonic to hypotonic, two-way diagonal electrophoresis of re-release type, as well as comparative study of re-release of two kinds of red blood cells (red blood cells in whole blood and those isolated from it) and the influence of plasma elements on the re-release of Hb in the red blood cells. These methods have provided certain bases for research into the existence states of in vivo intracellular proteins and opened up new approaches and new fields of research.