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Use of ion-exchange and hydrophobic-interaction chromatography for the rapid purification of lysozyme-estrone glucuronide conjugates

Smales, Christopher Mark, Cooke, D.G., Blackwell, Leonard F. (1994) Use of ion-exchange and hydrophobic-interaction chromatography for the rapid purification of lysozyme-estrone glucuronide conjugates. Journal of Chromatography B-Biomedical Applications, 662 (1). pp. 3-14. ISSN 0378-4347. (doi:10.1016/0378-4347(94)00381-5) (The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided) (KAR id:6201)

The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided.
Official URL:
https://doi.org/10.1016/0378-4347(94)00381-5

Abstract

Estrone glucuronide conjugates of hen egg white lysozyme were prepared by both the mixed-anhydride and active-ester coupling procedures. Both methods gave good yields of conjugate but the active-ester procedure gave a more diverse range of products consistent with a greater acylating ability. Unreacted lysozyme which was present in all cases was removed by a combination of cation-exchange chromatography on a Pharmacia Mono-S column and hydrophobic-interaction chromatography on an Alkyl Superose column. The conjugate families were more hydrophobic than native lysozyme. The chromatographic behaviour of the reaction mixtures on Mono S columns under non-denaturing conditions was complex as a result of hydrophobic effects and only at pH values above 7.0 did the conjugates elute in the order of their overall charges. At pH values below 6.0 the conjugates, although less charged than lysozyme, eluted last on salt gradients. In contrast when denaturing 7 M urea buffers were used the conjugates eluted in the order of their electrostatic charges and reproducible patterns were obtained which served as an excellent analytical system for lysozyme-steroid glucuronide conjugates. The purified conjugate material from the active-ester reaction gave over 90% inhibition of the lytic activity in the presence of an estrone glucuronide antibody. When used in a homogeneous enzyme immunoassay system the levels of urinary estrone glucuronide encountered in a normal menstrual cycle were easily measured.

Item Type: Article
DOI/Identification number: 10.1016/0378-4347(94)00381-5
Subjects: Q Science
Divisions: Divisions > Division of Natural Sciences > Biosciences
Depositing User: Mark Smales
Date Deposited: 01 Oct 2009 17:17 UTC
Last Modified: 09 Mar 2023 11:30 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/6201 (The current URI for this page, for reference purposes)

University of Kent Author Information

Smales, Christopher Mark.

Creator's ORCID: https://orcid.org/0000-0002-2762-4724
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