Abstract:
:Two methods employing a water-soluble carbodiimide for carboxyl activation were investigated for the immobilization of biochemicals to succinamidopropyl-porous glass beads. Immobilization using the simultaneous method (simultaneous addition of carbodiimide and nucleophilic ligand to the beads) and large excess of carbodiimide and a small nucleophile should result in covalent binding to all accessible carboxyl groups. Results obtained with glycine methyl ester indicated that 40% of the total surface carboxyl groups were sterically accessible. Using these reaction conditions with the protein, chymotrypsinogen, suggests that a surface monolayer is immobilized. although far fewer sites are required assuming single point attachment. For ligands containing carboxyl groups and several nucleophilic groups (e. g., enzymes), however, biological inactivation may occur using the simultaneous method. Consequently, a sequential method (activation of the surface with carbodiimide followed by washing and addition of the biochemical to be immobilized) was optimized. Using optimal conditions (20 min activation time at pH 4.75 and room temperature; 2 min wash at pH 7 and 0 degrees C) and 0.1M carbodiimide, nearly half of the accessible surface sites remained in the O-acylisourea form and reacted with glycine methyl ester upon its addition. The amount of surface loading as a function of activation time was consistent with a model constructed using rate constants for O-acylisourea formation and hydrolysis previously derived from solution studies with acetic acid [Swaisgood and Natake, J. Biochem 74, 77 (1973)]. Measurement of reaction rates with glycine methyl ester following surface activation suggests that the rate of reaction with amino groups is at least eightfold greater than the hydrolysis rate. Either immobilization procedure gave comparable enzyme loading and specific activities for the case of sulfhydryl oxidase.
journal_name
Biotechnol Bioengjournal_title
Biotechnology and bioengineeringauthors
Janolino VG,Swaisgood HEdoi
10.1002/bit.260240504subject
Has Abstractpub_date
1982-05-01 00:00:00pages
1069-80issue
5eissn
0006-3592issn
1097-0290journal_volume
24pub_type
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journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/bit.260370207
更新日期:1991-01-20 00:00:00
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journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
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更新日期:1983-08-01 00:00:00
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journal_title:Biotechnology and bioengineering
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pub_type: 杂志文章
doi:10.1002/bit.260300219
更新日期:1987-08-05 00:00:00
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journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
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journal_title:Biotechnology and bioengineering
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更新日期:2004-01-20 00:00:00