| High purity wheat germ agglutinin (WGA) was purified from wheat germ, and investigations were conducted on its anti-tumor activities, structural characteristics, and ligand binding properties.A convenient, sensitive, and reliable protocol was established to determine the hemagglutination activity (HA) of WGA which features sensitization and fixation of the effector red blood cells (RBC) respectively by trypsin digestion and glutaraldehyde crosslinking, utilization of pH7.2 isotonic phosphate buffered saline (PBS) as working media, replacement of test tubes conventionally used in Liener method by glass colorimetric cells of 0.5cm path-length, determination of end point to read the absorbance after 2h incubation, deduction of the contribution of the self-sedimetation of RBC, introduction of external standards to correct the results for variations between RBC batches and storage periods, and use of average of 5 parallel tests to calculate the HA of WGA. The routine thus established exhibited satisfactory sensitivity, and a standard deviation of 25% that of Liener method. The frequent need to re-prepare RBC suspensions in the case of natural RBC was also avoided.The preparation technique of WGA was improved and optimized. The extraction conditions was determined: extraction time 3.4h, formic acid concentration 0.5mol/L, and buffer/material ratio 10.15. WGA was then purified from the extract consecutively by use of salting-out techniques, selective heating treatment, affinity chromatography with partially hydrolyzed NAcGlc polymers, and gel permeation chromatography. The resulting WGA preparation showed a single band on SDS-PAGE, demonstrated same mobility as that of Sigma standard, and revealed a relative molecular weight in agreement with corresponding Brookhaven protein databank (PDB) records.Isolectin WGAII was purified from the mixture obtained as above by SP-Sephadex ion exchange chromatography, which showed same amino acid composition as the corresponding PDB records. In vitro experiments demonstrated inhibitive effects on the proliferation of two breast cancer cells, namely, MCF-7 and SHZ, both cases showing maximum inhibition at 72h after incubation. SHZ was especially sensitive to WGAII treatment, its LD50 being 10μg/ml. WGAII treatment crimpled, stretched, clustered, and detached the cancer cells, and worsens their surface reflectivity. The wall of some cells was blebbed, conglutinated, with some of the blebs broken or detached as apoptotic bodies. WGAII blocked the SHZ cell cycle at the G1/S phase, and thus interfered with the normal proliferation of cancer cells. FT-IR and Raman spectra demonstrated that WGAII, WGAI, and WGAIII powder lacked α-helices; and that the β-strand, β-turn and random coil content of WGAII was in between that of WGAI and WGAIII. Raman spectra also indicated that at saturation levels in an acidic solution, WGAII contained significant proportions of random coils and a certain amount of α-helices; WGAI contained a large amount of random coils, relatively more β-strands, and a certain fraction of α-helices; and WGAIII contained a large amount of of α-helices and random coils, and a certain fraction of β-strands. While at saturation levels in a neutral solution, WGAII contained a large amount of random coils and a remarkable amount of α-helices, and WGAI and WGAIII have remarkable amounts of random coils and α-helices. Raman spectra of the isolectin powders revealed that the Phe intensity of WGAII was between that of WGAI and WGAIII; that the degree of exposure of Tyr residues was about the same for WGAII, WGAI, and WGAIII, the exposed –OH groups of Tyr being completely exposed as both donors and receptors of hydrogen bonds; and that the Trp residues of WGAII and WGAIII were located in microenvironments of certain hydrophobicity, while that of WGAI in non-hydrophobic microenvironments and even came into contact with water molecules. At saturation levels in acidic solutions, WGAII and WGAIII exhibited no Phe signals, while WGAI showed Phe signals of...
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