The effects of isolation buffers on the properties of alpha-crystallin.
This work was undertaken in order to resolve some of the controversy in the literature concerning the properties of alpha-crystallins isolated in different laboratories. Bovine lens proteins were extracted and isolated by gel chromatography using 'Hoenders buffer' (0.02 M Tris-HCl, 1 mM EDTA, 80 mM NaCl, pH 7.3), 'Tardieu buffer' (0.04 M phosphate, 1 mM EDTA, 0.2 mM DTT, 0.06 M KCl, pH 6.8) and 'Thomson/Augusteyn' buffer (0.05 M Tris-HCl, 2 mM EDTA, 0.2 mM DTT, pH 8.0). The alpha-crystallin peaks were then divided into 12-16 pools and subjected to detailed physicochemical characterization. Fractionation by HPLC-GPC and quasi-elastic light scattering indicated that the size of the proteins decreased with increasing elution volume and that they were stable for at least 9 months at 20 degrees C. Molecular masses were found to range from over 2 mDa at the front of the peaks to around 600 kDa at the end. The size distributions, for the three buffers, were indistinguishable. No differences could be detected in the polypeptide distributions by SDS-PAGE. The proteins were also identical in their near- and far-UV circular dichroism spectra, accessibility of their sulphydryl groups to DTNB, tryptophan accessibility to quenching by acrylamide and iodide, and immunoreactivity with two monoclonal antibodies with different specificities. It is concluded that identical alpha-crystallins are isolated with the three different buffers and that variations in pH (6.9-8.0), ionic strength (60-150 mM) and cation (K, Na, Tris) during the isolation do not affect the properties of the protein. Claims that differing observations on the properties of alpha-crystallin may be attributed to the buffers used, are untenable.
Augusteyn
R C
RC
Russell Grimwade School of Biochemistry, University of Melbourne, Parkville, Victoria, Australia.
Parkhill
E M
EM
Stevens
A
A
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002021:Buffers; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003459:Crystallins / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D013438:Sulfhydryl Compounds / Q000032:analysis; D014364:Tryptophan / Q000032:analysis
D000911:Antibodies, Monoclonal; D002021:Buffers; D003459:Crystallins; D013438:Sulfhydryl Compounds; D014364:Tryptophan