Development of chromatographic bioseparations based on lectins and supermacroporous affinity cryogels

Abstract

Various cytomorphologic and biochemical markers of apoptosis are found in different compartments (plasma membrane, cytoplasm, nucleus, and mitochondria) of target cells. Although the plasma membrane is an easily accessible cellular compartment, relatively little is known about the changes in the expression of plasma membrane glycoproteins during apoptosis, and whether these changes could be used for detection of apoptosis. A critical element of this study was to purify lectins from crude homogenate on glycoprotein-cryogel affinity matrices, and later use the lectins to detect changes on the cell surface of apoptotic cells. Pterocarpus angolensis seed lectin was extracted and fractionated using ammonium sulphate precipitation. The 60 % ammonium sulphate pellet was dissolved in saline azide and purified using Sephadex G-75 affinity chromatography. A 28 kDa lectin was retarded within the column and appeared as a short and broad peak on the chromatogram. Traditionally, Sephadex G-75 column are used predominantly for size exclusion, in this study, the column was used in a non-traditional way for affinity chromatography, as the purified protein is able to bind sugar moieties existing in the structure of Sephadex G-75. A single-step purification of P. angolensis seed lectin was achieved by directly applying unclarified P. angolensis crude extract to the pAAm-cryogel using fetuin as the affinity ligand. Pterocarpus angolensis extract fractionated into 2 peaks, which revealed a highly concentrated band on SDS-PAGE. The results also revealed that an increased binding of the lectin to the fetuin-cryogel matrices was also dependent on the time of incubation. This study suggested very low capacities of the cryogels for the protein due to low coupling sites on the matrix. Taking into account that lectins serve as invaluable tools in diverse area of biomedical research, this study proposed using specific plant lectins to follow the expression of plasma membrane glycoproteins during programmed cell death. Treatment of HL-60 cells with lithium and actinomycin D confirmed a time- and dose-dependent inhibition of proliferation and a decrease in proliferation, which suggest cell death of the treated cells. The observed cell death was further investigated for cellular and biochemical hallmark features of apoptosis, which has shown preferential binding of annexin V-FITC to phosphatidylserine and low molecular DNA ladder. Several FITC labelled lectins were used to detect changes in cell surface glycosylation that accompany apoptosis. This study xvii has shown amongst several FITC-labelled lectins that T. vulgaris lectin could intensively stain the membrane area of apoptotic cells suggesting that the expression of N-acetylglucosamine was significantly increased during actinomycin D induced apoptosis of HL-60 cells. Binding was shown to be specific because it was blocked by the corresponding inhibitory sugar. Thus, the method described in this study could be suitable for the detection of very early stages of apoptosis by recognizing the cell surface carbohydrates of apoptosis.