Adsorption behavior of bovine serum albumin on lowly activated anionic exchangers suggests a new strategy for solid-phase proteomics.
Diluted solutions of bovine serum albumin (BSA) (e.g., 0.1 mg /mL) do not form detectable protein large aggregates. Using gel-filtration experiments, we determined that a diluted solution of BSA is 97% monomeric BSA and 3% dimeric. The adsorption of this diluted BSA on highly activated anionic exchangers (e,g., having 40 micromol/wet g) keeps this mainly monomeric form. When supports activated with 2 micromol/wet g are used, only dimers become adsorbed to the support, accounting for 100% of the offered BSA. When the diluted BSA solution is offered to very mildly activated anionic exchangers (even only 0.125 micromol/wet g), an unexpected adsorption of most of the BSA on the support was also observed. These very slightly activated supports are only able to adsorb very large proteins or very large protein-protein complexes, larger than BSA dimers. In fact, a rapid cross-linking of the adsorbed BSA with dextran-aldehyde reveals the formation of very large BSA-BSA complexes with molecular mass higher than 500 000 Da, complexes that may be observed for soluble BSA with very high concentrations but are not detectable at 0.1 mg/mL. Moreover, the size of the aggregates strongly depends on the concentration of the ionized groups on the support: the less activated the supports are, the higher the sizes of the complexes. It seems that the interaction of the BSA molecules on the margins of the BSA aggregate with the groups on the support may stabilize the whole protein aggregate, although some components are not interacting with the support. Aggregates could account for more than 40% of the BSA in the solution after 50 h of incubation. However, only these large BSA aggregates were adsorbed in the support.
Fuentes
Manuel
M
Departamento de Biocatalisis, Instituto de Catalisis, Consejo Superior de Investigaciones Científicas, Campus UAM, Cantoblanco, 28049 Madrid, Spain.
Pessela
Benebides C C
BC
Mateo
C
C
Palomo
Jose M
JM
Batalla
Pilar
P
Fernandez-Lafuente
Roberto
R
Guisan
Jose M
JM
D000327:Adsorption; D000818:Animals; D000838:Anions / Q000737:chemistry; D002417:Cattle; D002850:Chromatography, Gel / Q000379:methods; D007474:Ion Exchange; D008970:Molecular Weight; D040901:Proteomics; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D012995:Solubility; D012996:Solutions / Q000737:chemistry; D013499:Surface Properties; D013997:Time Factors
D000838:Anions; D012996:Solutions; D012710:Serum Albumin, Bovine