@article{c5c12861a05944429bb6cf95f1f7677e,
title = "Affinity chromatography of 3-oxosteroid Δ4-Δ5-isomerase of Pseudomonas testosteroni",
abstract = "Affinity chromatography of 3-oxosteroid Δ4-Δ5-isomerase (EC 5.3.3.1) of Pseudomonas testosteroni on 19-nortestosterone-17-O-succinyl diaminodipropylamino-agarose constitues a powerful and highly effective step in the purification of the enzyme in preparation for crystallization.",
author = "Benson, {Ann M.} and Suruda, {Anthony J.} and Roger Shaw and Paul Talalay",
note = "Funding Information: adsorbed as a clearly visible band near the top of the column, while retarded in this system, was gradually eluted by the 0.1 M Tri~phosphate. Subsequent elution with 1 M sodium acetate (pH 5.5) in 20 % glycerol yielded 71 mg of steroid A -isomerase (97 % recovery) with a specific activity of 38 100 units/mg (76 % pure), representing a 10.8-fold purification. The affinity chromatography was completed in less than 10 h, with flow rates of 150 ml/h during sample application and washing and 75 ml/h during elution of the enzyme in acetate-glycerol buffer. Following dialysis and concent~tion, the isomerase can easily be obtained in pure form by crystallization from ammonium sulfate solution [ 51. The specific activity of the crystalline isomerase is identical to that found previously for the pure enzyme [ 51. The extent of prior purification of the isomerase has been found to be an impo~ant factor with respect to both the capacity of the affinity adsorbent for the enzyme and the final specific activity attained in the chromatography. Isomerase of high specific activity has not been obtainable directly by affinity chromatography of crude extracts of P. testosteroni. However, affinity chromatography of the isomerase fractions from the DEAE-cellulose step (7-S % pure enzyme) is now a routine step in the large-scale preparation of crystalline steroid A -isomerase in this laboratory. The affinity chromatography step has certain distinct advantages. (I) It eliminates the tedious calcium phosphate gel chromatography procedure [ 51 and the time-consuming preparation of this adsorbent. (2) It can raise the specific activity of lower activity fractions than those here illustrated (e.g. DEAE-cellulose chromatography side fractions) although it is not suitable for direct purificatio1~ of crude initial extracts. (3) The no~estosteron~ agarose is highly stable, on storage at 4 “C for at least 2 years, and can be repeatedly regenerated after use by washing with 6 M guanidine hydrochloride [S] and reequilibrating with initial buffer. (4) The adsorbent gives highly reproducible chromatographic patterns, even after repeated uses. The authors wish to thank Dr Pedro Cuatrecasas and Dr Indu Parikh for helpful discussions concerning affinity chromatography, and to acknowledge the support of these studies by Grant AM 07422 from the National Institute of Arthritis and Metabolic Diseases of the National Institutes of Health and by the Gustavus and Louise Pfeiffer Foundation of New York. Copyright: Copyright 2014 Elsevier B.V., All rights reserved.",
year = "1974",
month = may,
day = "29",
doi = "10.1016/0005-2760(74)90245-8",
language = "English (US)",
volume = "348",
pages = "317--320",
journal = "Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism",
issn = "0005-2760",
publisher = "Elsevier",
number = "2",
}