Enhancing ligand-protein binding in affinity thermoprecipitation: elucidation of spacer effects

Vaidya, A. A. ; Lele, B. S. ; Kulkarni, M. G. ; Mashelkar, R. A. (1999) Enhancing ligand-protein binding in affinity thermoprecipitation: elucidation of spacer effects Biotechnology and Bioengineering, 64 (4). pp. 418-425. ISSN 0006-3592

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Official URL: http://www3.interscience.wiley.com/journal/7100346...

Related URL: http://dx.doi.org/10.1002/(SICI)1097-0290(19990820)

Abstract

Copolymers of N-isopropylacrylamide and N-acryloyl amino acid spacers of varying chain length were synthesized. p-Aminobenzamidine (PABA) was chemically linked to the pendant carboxyl groups of these polymers to obtain thermoprecipitating affinity polymers. The inhibition constant (Ki) of these polymers for trypsin decreased, i.e., the efficiency of PABA-trypsin binding increased with increase in the spacer chain length. The polymer to which PABA was linked through a spacer of five methylene groups exhibited eleven times lower Ki than that of the polymer containing PABA without a spacer. Investigations on model inhibitors N-acyl-p-aminobenzamidines showed that this enhancement in trypsin binding by the polymers was due to the spacer as well as to microenvironmental effects. Recovery and specific activity of the trypsin recovered increased with the spacer chain length. Separation of trypsin from a mixture of trypsin and chymotrypsin was also enhanced with the spacer chain length. The inhibition constants of these affinity polymers were not adversely affected by the crowding effect.

Item Type:Article
Source:Copyright of this article belongs to John Wiley and Sons, Inc.
Keywords:Affinity Precipitation; Spacers; p-aminobenzamidine; Trypsin
ID Code:17105
Deposited On:16 Nov 2010 08:29
Last Modified:04 Jun 2011 08:18

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