| Autor: |
Stolowitz, M. L., Ahlem, C., Hughes, K. A., Kaiser, R. J., Kesicki, E. A., Li, G., Lund, K. P., Torkelson, S. M., Wiley, J. P. |
| Zdroj: |
Bioconjugate Chemistry; March 21, 2001, Vol. 12 Issue: 2 p229-239, 11p |
| Abstrakt: |
A chemical affinity system exhibiting antibody-like properties is described. The system exploits bioconjugates with appended phenylboronic acid (PBA) moieties and a support-bound phenylboronic acid complexing reagent derived from salicylhydroxamic acid (SHA) for protein immobilization on a chromatographic support. The structure of the PBA·SHA complex was characterized by 11B NMR and mass spectrometry and compared with complexes derived from model compounds. Protein modification reagents were synthesized from 3-aminophenylboronic acid and utilized to prepare bioconjugates from alkaline phosphatase (AP) and horseradish peroxidase (HRP). AP obtained from one source afforded PBA bioconjugates exhibiting significant loss of enzymatic activity, whereas AP obtained from a second source afforded PBA bioconjugates exhibiting only a modest loss of enzymatic activity. Conversely, HRP afforded PBA bioconjugates exhibiting no loss of enzymatic activity. SHA-modified Sepharose was prepared by reaction of methyl 4-[(6-aminohexanoylamino)methyl]salicylate with CNBr-activated Sepharose 4B, followed by treatment with aqueous alkaline hydroxylamine. PBA−AP and PBA−HRP conjugates were efficiently immobilized on SHA−Sepharose at pH 8.3. PBA−AP conjugates were retained after washing with acidic buffers at pH 6.7, 4.2, and 2.5, whereas PBA−HRP conjugates were retained after washing with buffer at pH 6.7, but were eluted to some extent at and below pH 4.2. The results are interpreted in terms of multivalent interactions involving boronic acid complex formation between the enzyme bioconjugates and immobilized complexing reagent. |
| Databáze: |
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