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Enhancing ligand–protein binding in affinity thermoprecipitation: Elucidation of spacer effects
Author(s) -
Vaidya A. A.,
Lele B. S.,
Kulkarni M. G.,
Mashelkar R. A.
Publication year - 1999
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(19990820)64:4<418::aid-bit4>3.0.co;2-y
Subject(s) - trypsin , chemistry , polymer , chymotrypsin , ligand (biochemistry) , copolymer , stereochemistry , polymer chemistry , enzyme , organic chemistry , biochemistry , receptor
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 ( K i ) 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 K i 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. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 418–425, 1999.