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Multipoint binding in metal-affinity chromatography: II. Effect of pH and imidazole on chromatographic retention of engineered histidine-containing cytochromes c

Johnson, Robert D. and Todd, Robert J. and Arnold, Frances H. (1996) Multipoint binding in metal-affinity chromatography: II. Effect of pH and imidazole on chromatographic retention of engineered histidine-containing cytochromes c. Journal of Chromatography A, 725 (2). pp. 225-235. ISSN 0021-9673. http://resolver.caltech.edu/CaltechAUTHORS:20180823-144311160

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Abstract

Protein binding in immobilized metal affinity chromatography (IMAC) was studied using a set of Saccharomyces cerevisiae iso-1-cytochrome c variants which differed only in their histidine content and placement. Elution with an imidazole gradient enabled separation of cytochrome c variants based on their histidine multiplicity. Millimolar concentrations of imidazole dramatically decreased protein partitioning to the IMAC support as measured by the chromatographic capacity factors under isocratic conditions. Fitting the partitioning data to the “stoichiometric displacement” model indicates that cytochrome c variants containing from one to four surface histidines each displaced approximately three equivalents of imidazole upon adsorption. Therefore even a protein with a single surface histidine appears to coordinate to multiple copper sites on the IMAC support at neutral pH. The effect of pH on the capacity factors of these variants measured in the absence of imidazole further supports this interpretation. Although the presence of a surface histidine was required for retention at neutral pH, a variant with no surface histidines still partitioned strongly to the IMAC support at higher pH (pH > 7.5). These results indicate the contribution of additional protein-metal-coordinating groups, presumably surface amines, to chromatographic retention in IMAC.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/0021-9673(95)00992-2DOIArticle
ORCID:
AuthorORCID
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 1996 Published by Elsevier. Received 11 July 1995, Revised 1 September 1995, Accepted 1 September 1995. This research is supported by the National Science Foundation. F.H.A. acknowledges an NSF PYI award and a fellowship from the David and Lucile Packard Foundation. R.D.J. and R.J.T. acknowledge predoctoral training fellowships from the U.S. National Institute of General Medical Sciences, Pharmacology Sciences Program.
Funders:
Funding AgencyGrant Number
NSFUNSPECIFIED
David and Lucile Packard FoundationUNSPECIFIED
NIH Predoctoral FellowshipUNSPECIFIED
Subject Keywords:Immobilized metal-affinity chromatography; Histidine-metal interactions; Protein-metal interactions; Retention mechanisms; Cytochrome c; Imidazole; Histidine; Proteins
Record Number:CaltechAUTHORS:20180823-144311160
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20180823-144311160
Official Citation:Robert D. Johnson, Robert J. Todd, Frances H. Arnold, Multipoint binding in metal-affinity chromatography II. Effect of pH and imidazole on chromatographic retention of engineered histidine-containing cytochromes c, Journal of Chromatography A, Volume 725, Issue 2, 1996, Pages 225-235, ISSN 0021-9673, https://doi.org/10.1016/0021-9673(95)00992-2. (http://www.sciencedirect.com/science/article/pii/0021967395009922)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:89106
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:23 Aug 2018 22:31
Last Modified:23 Aug 2018 22:31

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