A. D. Landman a n d N. N. Landman Faculty of Dentistry University of Manitoba Winnipeg, Canada R3E OW3
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The Binding of Biotin to SepharoseAvidin Column Demonstration of the affinity chromatography technique
The publication of several comprehensive reviews on affmity chromatography ( 1 3 )and, in particular, the additional volume in "Methods in Enzymology" series devoted to the subject ( 4 ) ,have further established this technique as a major conventional procedure in preparative and analytical biochemistry. The concept of affinity chromatography was first introduced in 1953 by Lerman (5), who demonstrated the binding of aspecific enzyme to a cellulose column into which the appropriate substrate was embedded. The intensive use of the technique was delayed, however, for a decade until convenient methods for attaching ligands to support gels were available. In 1967 it was shown by Axen, Porath, and Ernhack (6) that when a Sepharose gel is mixed with cyanogen bromide a t p H 10-11, the gel is activated and will attach covalently organic molecules which have free amino groups. The wide use of the technique was further facilitated after other methods for the attachment of ligands to support matrices such as polyacrylamide heads, styrene-divinylhenzene beads and porous glass granules were established. While ion exchangers and molecular sieves will separate protein species on the basis of their charge and size, an affinity column is highly specific and can be designed to isolate a single protein. Cuatrecasas and Wilchek (7) used a biocytin-Sepharose column for a single step purification of avidin, a glycoprotein found in egg white. Other investigators used avidin attached to a Sepharose matrix in order to hind biotin containing proteins (8-10). Indeed, the binding between avidin and biotin, a vitamin found in biological samples in minute amounts, can he utilized very effectively for affinity chromatography. It is particularly suitable for the demonstration of the concept and the methodology of this technique in a biochemistry laboratory course. Materials Cyauogen bromide activated Sepharose is the product of Pharmacia Fine Chemicals, Uppsala, Sweden. D-(carhonyl 14C)hiotin 46 mCi/mmole and Aquasol are available from New England Nuclear, Canada. Avidin 12 unitslmg, unlahelled hiotin, bovine serum albumin (BSA), hemoglobin, riboflavin, and [2,(4-hydroxyazobenzene)henzoic acid] (HABA) were purchased from Sigma Chemical Company, St. Louis, Mo. The Experimental Procedure
Attachment of Avidin to Activated Sepharose
The covalent binding of the protein to the gel is performed according to a procedure recommended by the manufacturer (3).One gram of the freeze dried activated Sepharose powder is washed with 200 ml of 1mM HCI on a small sintered glass funnel. The washed gel is mixed with 2 mg of avidin dissolved in 10 ml of 0.1 M NaHCOs, pH 8.3, containing 0.5 M NaC1, and the mixture is then shaken for 2 hr st room temperature. The coupling is accomplished at this step and the excess of the activated Sepharose moieties,which did not interact with avidin, are blocked by repeating the procedure with 100 mg of BSA (9). The unbound BSA is washed away with 200 ml of the hicarbonate bufferthrough a sintered glass funnel, and the gel is then poured into a column made of a Pasteur pipet which has a tip loosely packed with glass wool. The Sepharose-avidin affinity column is stable at 4-8°C for at least a month, and therefore can be prepared at aperiod separate from the procedure described in the following section.
Demonstration of Column Specificity
Hemoglobin (1 ml, 500 ~ g l m l )riboflavin , (1 ml, 100 pgfml), and HABA (1 ml, 200 ~ g l m l are ) eluted sequentially through the Sepharose-avidin column with the bicarbonate-NaC1 buffer. Due to the small size of the column the elution is fairly fast, although the three substances may he slightly retarded by the Sepharose matrix, which acts as a molecular sieve. The elution can be followed visually since the protein and the dyes are stronelv " ,colored. HARA binds T Othe attached avidin sto~rhiometrically,and the of unbound dve is further eluted and discarded The . rxrecs -~ binding of HABA to the protein gives the'column a slight oranee tint. Biotin (1ml. 1uCi in 200 .rralml) - releases the HABA from the column and binds to the avidin in its place (8). HABA is a dve which binds to a varietv of proteins nonspeits hinding to avidin-is inproximity to the cifically. site a t which the protein binds to biotin, and when competing, the dye will be displaced by the vitamin. The sodium chloride in the buffer. added to minimize protein-protein interactions during the coupling of avidin t o t h e matrix (3), is advantageous for the binding of hiotin and avidin, since the affinity of the two cornpone& is a function of the ionic strength. The biotin is now released from the column by elution with 6 N guanidine-HC1, p H 1.5 (7,9). At this step the effluent is collected in 2-ml fractions into scintillation vials and 15 ml of Aquasol is then added. The labelled hiotin is counted in a liquid scintillation spectrometer. By definition one unit of avidin hinds one fig of biotin. From the specific activity of the labelled hiotin and the radioactivity released by guanidine, the amount of hiotin bound to the column can be calculated in moles. This is a direct indication of the amount of avidin bound to the affinity column and to the efficiency of coupling of the protein to the matrix (3,8). ~
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Discussion Some of the requirements of an efficient affinity column were outlined by Cuatrecasas and Anfinsen (I,2). These are summarized as follows I I There is a n esrenrinl rquiremenr ior n stnmg a f f i n ~ t ?heturen thr lignnd attached
