Weak Affinity Chromatography : Evaluation of Different Silica Supports for Protein Immobilizationand Effect of Mobile Phases Regarding Retentionand Non-specific Binding

• Main Author: Westerhult, David
• Format: Others
• Language: English
• Published: Linnéuniversitetet, Institutionen för naturvetenskap, NV 2012
• Subjects: Chemistry; Kemi
• Online Access: http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-17968

Fragment based lead discovery (FBLD), where libraries of small fragments are screened andlater on developed to lead compounds, is an alternative to the classical drug discovery methods such as high trough-put screening. Weak affinity chromatography (WAC) is a new promising approach to the screening process of FBLD. WAC is performed by injections of fragments onto a high performance liquid chromatography (HPLC) column in which a protein is immobilized to a silica support. The retention of the injected fragments is correlated to the binding affinity of the fragments towards the immobilized protein. Immobilization capacity of three different silica materials with varying pore size (Kromasil240 Å, Nucleosil 1000 Å and Kromasil 300 Å) was evaluated by immobilization of trypsin. Retention of benzamidines on the trypsin columns was evaluated with different mobile phases. Contribution of non-specific binding in the interaction between the 4-aminobenzamidine and thrombin was estimated by frontal chromatography on a capillary columnusing PBS and PBS/acetonitrile as mobile phases. This study showed that the Kromasil 300 Å had a superior immobilization capacity of trypsin compared to the Kromasil 240 Å andthe Nucleosil 1000 Å (100 mg compared to 87.4 mg and 15.1 mg trypsin/g silica, respectively). However, the Nucleosil 1000 Å might be a more suitable support for the immobilization of larger proteins. Adding 5 % methanol or acetonitrile to the mobile phase resulted in a significant (p < 0.05) decreased retention of benzamidine fragments on the trypsin 240 Å column. Non-specific binding between thrombin and 4-ABA was not statistically significantly altered when 5 % acetonitrile was added to the mobile phase.