Study On Purification, Characterization And Conformation Of Agrocybe Cylindracea Lectin

The study on Agrocybe cylindracea Lectin(ACL) main include the purification and characterization of ACL, the relationship between structure and bioactivity of ACL.ACL was isolated from isolated and characterized a lectin from the fruiting bodies of the edible mushroom Agrocybe cylindracea, by three-step procedure consisting of ion exchange chromatography in a DEAE-Sepharose colum, Sephacryl S-100 and CM-Sepharose. The lectin was found to possess two subunits with a small difference in molecular weight (16.1 kDa and 15.3 kDa). The molecular weight was 31.2 kDa determined by gel filtration on Sephacryl S-100.ACL can agglutinate rabbit erythrocytes, human blood type A, B, AB, O. the lectin agglutinated all of these blood cells showing no human blood specificity; however, it was distinctly characterized by its slight hemagglutinating activity toward the A blood group.The fluorescence spectrum of ACL excited at 280 nm showed a maximum peak at 337nm. The characteristic peak of Tyr did not exist, and it showed that the fluorescence energy of Tyr was transformed to Trp and strength the fluorescence of Trp. When ACL was excited at 295nm, the fluorescence spectrums show a maximum peak at 337nm, the λ^max of fluorescence emission spectrum blue-shifted more than 10nm compared with the λ^max of free Tyr (348nm). Fluorescence studies of ACL indicated that Trp residues were present in a highly hydrophobic environment.ACL was modified with N-Bromosuccinimide (NBS) in HAc-NaAc buffer containing 8mol/L urea or not. The result indicated 2.09 Trp was modified in the buffer with urea, and one of them was essential group to the agglutinate activity of ACL. Sulfhydryl groups were modified by N-ethymaleimide (NEM), there was one -SH in a ACL molecule. The other groups modified such as Arg, Tyr, Ser, His did not affect the hemagglutinating activity of ACL. The result indicated the Trp residues were essential to the hemagglutinating activity and were involved in carbohydrated-binding site.ACL lost its agglutinate activity after removing the metal ions of ACL with EDTA. Incubated the deionized ACL with Ca2\ Mn2+> Mg2\ Zn2+respectively, Agglutinate activity of ACL recovered near to the level of natural ACL with Ca2+ and Zn2+. The series of trials indicated some ions were helpful to stabilize of the configuration of ACLACL was little sensitive to pH and totally lost its activity when incubated with all pH values below pH 4 and above pH 11. At 50 °C, 60 °C ACL was fairly stable; However when heated at 80 °C, it lost almost its original activity.The equilibrium denaturation of ACL in urea, guanidine hydrochloride (GdnHCl), SDS, 0 -me, iodoacetic acid has been examined by steady-state fluorescence. The urea and guanidine hydrochloride (GdnHCl) of ACL reveals two distinct and separable transitions: dissociation and unfolding. The SDS denaturation made ACL lose its original activity. But P -me and iodoacetic acid effect ACL little.The study on fluorescence quenching showed that the fluorescence from Trp residues were quenched by KI, CsCl and acrylamide. 87.7% of Trp were quenched by acrylamide, 79.8% of Trp were quenched by KI, and CsCl quenched 51.54% of Trp. It means that most of Trp residues of ACL were located on hydrophilic surface, and the amount of Trp residues on hydrophilic surface and inside were almost equal.