Selective and covalent immobilization of proteins onto polymeric surfaces. biotechnological applications

CARBAJAL, M.L.; SANTOS, J.; ERMACORA, M.R.; GRASSELLI, M

Bariloche, Río Negro, Argentina.

Congreso; XXXIX Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIBBM); 2003

SAIBBM

Covalent chemical immobilization is an important field of biochemistry and physic chemistry. Also, it is a useful tool in modern biotechnology to develop heterogeneous systems for diagnostic kits. Carbodiimide coupling reaction is one of the most used as a consequence of its very well known mechanism and reaction conditions. However, the high abundance of carboxylic acids and amino groups in protein structures renders this reaction unspecific. In order to study less explored chemical reactions for protein immobilization, sulphur and arsenic containing compounds were reinvestigated. The chemistry of these compounds has convenient features: they form covalent bonds that are reversible under mild conditions and involve short reaction times. In this work, reactions of aminophenyl arsine oxide (APA3), aminophenyl arsine chloride (APA3Cl2), and aminoarsalinic acid (APA5) with biological sulfhidryl compounds were studied in solution. Reaction products were analysed by UV, HPLC and MALDI-MS. As expected, APA3 reacted readily with mercaptoethanol and dithiotreitol. Interestingly, APA3Cl2 showed higher solubility than APA3 and similar rates of reaction with the thiol compounds. Natural or mutant proteins showing Cys-X-X-Cys motive in their primary structure could be immobilized onto polymeric surfaces containing APA3. Additionally it was possible to selectively immobilize these proteins from a crude extract without previous purification.