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Isolation and biophysical studies of horse plasma gelsolin

University of British Columbia

Gelsolin from horse plasma has been isolated in good and reproducible yields. It has an absorption coefficient of 1.4 ml/(mg cm) and is similar in amino acid composition to other plasma gelsolins. It migrates as a single polypeptide chain in polyacrylamide gel electrophoresis with an apparent molecular mass of 90 kDa. Hydrodynamic calculations suggest that gelsolin is a globular protein of 75 kDa. Thermal and chemical denaturation profiles were obtained for gelsolin by measuring its intrinsic fluorescence and ellipticity values. The melting temperature values obtained were Tm ≈ 46 °C in the absence of calcium and Tm ≈ 54 °C in the presence of the divalent cation. The mid-point of the transition in the guanidine hydrochloride-induced unfolding of gelsolin was found to be at ≈ 1.5 M denaturant. Gelsolin is able to nucleate actin polymerization and sever actin filaments. These activities are manifested by the abolition of the lag phase in the time course of actin polymerization and the decreased steady state viscosity of actin solutions polymerized in the presence of gelsolin. Gelsolin interacts with actin labelled with the fluorescent probe 6-acryloyl-2-dimethylaminonaphthalene (acrylodan) to produce a 2:1 actin:gelsolin complex in the presence of calcium. Upon chelation of the divalent cation from the 2:1 complex, one actin molecule is released producing a 1:1 EGTA-resistant complex . The fluorescence of 2-(N-methylanilino)naphthalene-6-sulphonate (MANS), a fluorescent probe that is sensitive to the polarity of its environment, is both enhanced and blue shifted upon binding to gelsolin. These results are indicative of the binding of MANS to hydrophobic regions in gelsolin. Gelsolin binds 2.5 ± 0.9 molecules of MANS with a dissociation constant of 0.24 ± 0.13 μM. Gelsolin can be labelled in the absence of divalent cations with the sulfhydryl-specific probe N-(l-pyrenyl)iodoacetamide (PIA) without altering its structural integrity. The labelled protein presents excimer-like pyrene emission indicative of the proximity of the labelled cysteines in the three dimensional structure of the protein. The spectroscopic characteristics of the labelled protein indicate that the excimer emission arises from interactions that can be traced to the ground state of the pyrene molecules. This is in contrast to the well studied excimer emission that arises from molecules that repel each other in the ground state.

Text

2011-02-17

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http://hdl.handle.net/2429/31413

Chemistry

University of British Columbia

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