JOURNAL OF COLLOID AND INTERFACE SCIENCE 185, 313–316 (1997) ARTICLE NO. CS964566 Use of Chemically Modified Silica with b-Diketoamine Groups for Separation of a-Lactoalbumin from Bovine Milk Whey by Affinity Chromatography ALESSANDRA GAMBERO,* LAURO T. KUBOTA,² ,1 YOSHITAKA GUSHIKEM,² C LA ´ UDIO AIROLDI,² JOSE ´ M. GRANJEIRO,‡ EULA ´ ZIO M. TAGA,‡ AND EDE ´ SIO F. C. ALCA ˆ NTARA§ * Departamento de Quı ´mica, F.C. Unesp, Caixa Postal 473, 17033-360 Bauru, S.P., Brazil; ² Instituto de Quı ´mica, Unicamp, Caixa Postal 6154, 13083-970 Campinas, S.P., Brazil; ‡ Departamento de Bioquı ´mica, Faculdade de Odontologia, USP, Bauru, S.P., Brazil; and § Departamento de Quı ´mica, UFG, Goia ˆnia, Go, Brazil Received January 16, 1996; accepted August 22, 1996 area. Generally, organic matrices like agarose, cellulose, and Silica gel surface was chemically modified with b-diketoamine composite polymers have been used (4). groups by reacting the silanol from the silica surface with 3- Recent studies demonstrated that some mineral solids may aminopropyl-triethoxysilane and 3-bromopentanedione. With this be used as adsorbents of proteins. The surfaces of these material, copperions were adsorbed from aqueous solutions. The materials are highly developed and permit the immobiliza- chemical analysis of the silica-gel-immobilized acetylacetone pro- tion of functional groups. Among these solids, silica gel may vided a quantity of 0.67 mmol g 01 of organic groups attached to be used to this aim. The silica gel surface is covered with the support and 0.63 mmol g 01 of copper. This material was used silanol groups (Si–OH), which determine its chemical be- as a stationary phase in IMAC (immobilized metal affinity chro- havior (5–11). matography), to separate a-lactoalbumin from bovine milk whey. The results showed an efficient separation in the chromatographic The use of silica gel as a matrix for the immobilization column. The possibility of reutilization of the stationary phase was of molecules with organofunctional groups has some advan- also investigated.  1997 Academic Press tages over organic matrices, such as high surface area, a Key Words: modified silica;affinity chromatography;separation; narrow average pore size distribution, mechanical resistance, lactoalbumin. and rigidity (5, 11–13). Organofunctionalized silicas have found promising use as stationary phases in chromatographic analysis ( 14, 15 ) , as supports for enzyme immobilization in biotechnologi- INTRODUCTION cal applications (16, 17), and for adsorption of metallic ions (18, 19). In the recent advances of biotechnology, much emphasis In the present work the use of silica gel modified with has been given to techniques for the extraction, purification, diketoamine groups as a stationary phase for affinity chroma- and characterization of proteins (1). tography in a-lactoalbumin purification is described. Generally, the majority of these processes take a long time and involve various steps such as extract purification and EXPERIMENTAL fractionation, by physical methods, by using an ion-ex- change chromatography technique, or by affinity gel filtra- Extraction of the Milk Whey tion, among others (2). Affinity chromatography has been used successfully in The milk whey was extracted by centrifuging 10 ml of protein purification procedures, exploiting the biological natural bovine milk at 7000 rpm for 1 h. The supernatant, characteristics of the target protein that binds to small bio- 8.5 ml, was collected and the pH was adjusted to 4.6 with molecules (ligands) which are covalently attached to an in- a HCl solution in order to precipitate casein. The mixture soluble matrix (3). This method provides a 1000-fold pro- was submitted to constant agitation at 40°C for 30 min. After tein purification in a single step. this treatment, the mixture was centrifuged again at 7000 The ideal support for affinity chromatography should pres- rpm for 30 min and the precipitate was then discarded. In ent characteristics such as rigidity, stability, and high surface the following step the pH of the supernatant was adjusted to 6.0 with Tris buffer solution and then filtered. About 7.0 1 To whom correspondence should be addressed. ml of whey was obtained. 313 0021-9797/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.