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Multiple-site binding interactions in metal-affinity chromatography: I. Equilibrium binding of engineered histidine-containing cytochromes c

Todd, Robert J. and Johnson, Robert D. and Arnold, Frances H. (1994) Multiple-site binding interactions in metal-affinity chromatography: I. Equilibrium binding of engineered histidine-containing cytochromes c. Journal of Chromatography A, 662 (1). pp. 13-26. ISSN 0021-9673. https://resolver.caltech.edu/CaltechAUTHORS:20180823-144311357

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Abstract

Mechanisms of protein retention in immobilized metal-affinity chromatography (IMAC) have been probed using a set of Saccharomyces cerevisiae iso-1-cytochrome c histidine variants constructed by site-directed mutagenesis. Proteins containing a single accessible histidine exhibit Langmuir-type isotherms with maximum protein binding capacities between 5 and 10% of the maximum copper loading and the capacity of the support to bind imidazole. A simple model that assumes that the copper sites are densely packed and can be blocked by protein adsorption yields binding constants for single-histidine proteins that are similar to the binding constant for free imidazole. Proteins containing multiple accessible histidines do not exhibit simple Langmuir-type behavior; they appear to interact with the support by simultaneous coordination to more than one metal ion, the result of which is to increase the apparent binding affinity by as much as a factor of 1000. The protein binding constant depends on the availability of copper sites: binding is significantly weaker at low surface concentrations of copper that presumably cannot support multiple-site interactions. The protein binding capacity drops to zero at copper loadings less than one-half the maximum, indicating that immobilized iminodiacetic acid ligands are sufficiently close together that two can coordinate a single copper ion, which precludes its interaction with a protein. Protein adsorption via multiple-site coordination has important consequences for the optimization of IMAC separations and the design of new IMAC supports.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/0021-9673(94)85291-xDOIArticle
ORCID:
AuthorORCID
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 1994 Published by Elsevier. Received 16 August 1993, Revised 18 October 1993. This research is supported by the National Science Foundation (BCS-9108502) and the Office of Naval research (N00014-92-J-1178). F.H.A. acknowledges a fellowship from the David and Lucile Packard Foundation and a Presidential Young Investigator Award from the National Science Foundation. R.J.T. acknowledges a predoctoral training fellowship in biotechnology from the National Institute of General Medical Sciences, National Research Service Award 1 T32 GM 08346-01 from the Pharmacology Sciences Program. R.D.J. acknowledges a graduate fellowship from Kelco Division of Merck and Co., Inc.
Funders:
Funding AgencyGrant Number
NSFBCS-9108502
Office of Naval Research (ONR)N00014-92-J-1178
David and Lucile Packard FoundationUNSPECIFIED
NIH Predoctoral Fellowship1 T32 GM 08346-01
Merck and CompanyUNSPECIFIED
Issue or Number:1
Record Number:CaltechAUTHORS:20180823-144311357
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20180823-144311357
Official Citation:Robert J. Todd, Robert D. Johnson, Frances H. Arnold, Multiple-site binding interactions in metal-affinity chromatography: I. Equilibrium binding of engineered histidine-containing cytochromes c, Journal of Chromatography A, Volume 662, Issue 1, 1994, Pages 13-26, ISSN 0021-9673, https://doi.org/10.1016/0021-9673(94)85291-X. (http://www.sciencedirect.com/science/article/pii/002196739485291X)
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ID Code:89108
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:23 Aug 2018 22:33
Last Modified:03 Oct 2019 20:13

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