A micromethod for delipidation of aqueous proteins.
A simple and nondestructive method was developed for effective removal of lipids, such as long-chain fatty acids and steroids, from small quantities (2-10 mg) of aqueous protein. The procedure operates with a high recovery of protein (97%) and was elaborated by using different albumin preparations as model proteins. Delipidation was monitored by using [14C]palmitate or [3H]progesterone or by using an enzymatic method for quantitative determination of fatty acids. The essential feature of the method is a pH-induced (e.g., pH 3.0 or 12.5), partial unfolding of the protein which makes it possible for hydroxyalkoxypropyl derivatives of dextran to take over the lipids. In practice, a test tube containing an aqueous suspension of protein and dextran derivative of the desired pH is shaken or rotated carefully for a certain period of time. Afterward, the purified protein and the dextran are separated by applying the suspension on a small glass column equipped with a glass filter which allows for passage of the protein. The procedure was optimized with respect to dextran to protein ratio and volume of suspension in addition to pH and time. Furthermore, the effect of temperature on delipidation was examined. Finally, the possibility of using hydroxyalkoxypropyl derivatives of dextran or sephadex in radiochemical assays of lipid binding is discussed in detail.
Kragh-Hansen
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Institute of Medical Biochemistry, University of Aarhus, Denmark.
D000418:Albumins / Q000302:isolation & purification / Q000378:metabolism; D003911:Dextrans / Q000378:metabolism; D005227:Fatty Acids / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D050356:Lipid Metabolism; D008055:Lipids / Q000302:isolation & purification*; D008832:Microchemistry / Q000379:methods; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012685:Sepharose / Q000378:metabolism; D012995:Solubility; D013256:Steroids / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014867:Water
D000418:Albumins; D003911:Dextrans; D005227:Fatty Acids; D008055:Lipids; D011506:Proteins; D013256:Steroids; D014867:Water; D012685:Sepharose