Paul Madden

Abstract

In this thesis static light scattering was used to systematically examine potential protocols to effectively isolate bovine eye lens alpha crystallin subunits, to enable confident reconstitution and study of homogeneous assemblies of subunits. The light scattering properties of alpha crystallin subunits that were separated from one another by anion-exchange chromatography in urea, and subsequently reconstituted in buffer that did not contain urea were also studied. Such reconstituted assemblies may prove useful for future work examining the dependence of interactions between gamma and alpha crystallin on alpha crystallin subunit composition, the properties of high concentration gamma-alpha mixtures and other mixtures, and possibly the preparation of crystals for xray crystallography. The light scattering data yielded a molecular weight of (5.900 +/- 0.005) x 105 g/mole for the starting, native bovine alpha crystallin protein. The dimensionless virial coefficient of the native alpha crystallin was 3.1, less than the hard-sphere value, 4, and possibly consistent with attractive interactions on top of hard-core repulsion. While light scattering showed that 6 M urea did not completely disassemble alpha, the molecular weight after incubation in 8 M urea was (2.0 +/- 0.5) x 104 g/mole, in excellent agreement with the known molecular weights calculated from the sequences of bovine alphaA and alphaB. Reconstituted subunits isolated by anion exchange chromatography, believed to be alphaA crystallin, pending further confirmation, assembled to form particles with a molecular weight of (5 +/- 3) x 104 g/mol. These results set the stage for further study of the lens protein interactions involving homogeneous alpha crystallin assemblies.

Library of Congress Subject Headings

Crystalline lens; Cataract--Research; Calves--Diseases--Research; Light--Scattering

Publication Date

2-1-2010

Document Type

Thesis

Advisor

Not listed

Comments

Note: imported from RIT’s Digital Media Library running on DSpace to RIT Scholar Works in December 2013. Physical copy available through RIT's The Wallace Library at: QL949 .M34 2010

Campus

RIT – Main Campus

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