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Purification and Partial Characterization of a Low-molecular Trypsin Inhibitor from Oilrape

Author: LiaoHai
Tutor: DuLinFang
School: Sichuan University
Course: Biochemistry and Molecular Biology
Keywords: Cole Trypsin inhibitor Isolation and Purification Fluorescence analysis
CLC: TQ452
Type: Master's thesis
Year: 2002
Downloads: 256
Quote: 4
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Abstract


Protease inhibitors are a class can selectively reduce the activity of the target protease of the smaller molecular weight proteins. Protease inhibitor involved in the regulation of endogenous protease activity in the plant, plant storage protein transport, erosion protection of plants from fungi and insects, as new types of drugs used in the treatment of diseases such as cancer, AIDS has value, as the insect-resistant factor , has been widely used in the breeding of insect-resistant transgenic plants. The papers from the rape seed trypsin inhibitor purified from a part of the nature of research. Cruciferous plants; vegetables (Brassica napus var.) Seed pestle, after extracted with ether degreasing, sulfuric acid, 65 ° C heat denaturation impurity protein ,35-75% saturation ammonium sulfate segment salting crude extract was prepared. Electrophoresis activity staining detection methods, using the established the presence of two different molecular weight trypsin inhibitor (BNTIa, BNTIb) found in the crude extracts. Reductant processing experiments show that the destruction of the disulfide bonds will change the inhibitory activity and proved that the inhibitory activity of the disulfide thereof is necessary. Subjected to electrophoresis, under different pH conditions to determine their isoelectric points are 5.20. Further use of the DEAE-52 cellulose ion exchange column chromatography, wherein one kind trypsin inhibitor (BNTIa) of isolated and purified from the crude extracts obtained. SDS-PAGE analysis of their approximate molecular weight of 8700, containing chain disulfide bond, the activity staining of non-denaturing electrophoresis the apparent molecular weight of 35kD, indicating that BNTIa in the natural state may exist in the form of tetramer. The thermal stability experiments show BNTIa can be kept below 60 ℃ inhibitory activity and having a good thermal stability. BNTIa having 268nm of maximum absorption, showed BNTIa phenylalanine, tyrosine, tryptophan residues less. Protein fluorescence to detect conformational changes BNTIa different solution. When the wavelength of the excitation light of 280nm, natural BNTIa the maximum emission wavelength of 320nm, relative 303nm tyrosine emission peak appears redshift, showed that the tyrosine residue is located in the surface of the protein molecule, in a polar environment. In 80 ℃ the thermal denaturation 60min after, the maximum fluorescence emission peak blue shift of the fluorescence intensity decreased. 8mol / L urea treatment, the maximum emission peak is blue-shifted fluorescence intensity decreased. After lmmol / L dithiothreitol treatment, two emission peaks, the decrease in fluorescence intensity. 6 mol / L guanidine chloride treatment, the maximum emission wavelength red shift, the increase in fluorescence intensity. When an excitation wavelength of 295 urn, natural BNTIa the maximum emission wavelength of 340 urn, the relative 348 urn tryptophan emission peaks blue shift, indicating that the tryptophan residues are located inside the egg / white matter molecules. 80OC thermal denaturation for 60 min, the maximum emission peak red shift, the fluorescence intensity decreased. smol / L urea, the maximum emission peak red shift, the fluorescence intensity decreased. 6mol / L hydrochloric acid arc treatment, the red-shifted emission peak increase in fluorescence intensity. And lmmol / L dithiothreitol treatment, the most in emission peak blue shift, the fluorescence intensity decreased. The fluorescence analysis showed: disulfide bonds, hydrophobic interaction, van der Waals forces, hydrogen bonds and electrostatic interaction plays an important role in the structure of the sustain BNTIa

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