ANALYTICAL
BIOCHEMISTRY
71, 322-323
(1976)
A Simple Method to Introduce Aldehydic Function to Agarose Agarose has been widely used as a matrix in affinity chromatography and for immobilization of macromolecules. Many chemical methods to prepare these derivatives of agarose have been described (1). Aldehydes react in mild conditions with primary amines to form Schiff bases and thus gels containing aldehyde groups are proper intermediates in immobilizing ligands. Aldehydic function can be well introduced straight into the carbohydrate chains of agarose by sodium metaperiodate oxidation (1). However, this method of synthesis is not convenient when one wants to attach a ligand to the aldehyde group at the end of a spacer. The use of alkylamino support and glutardialdehyde (2) suffers from the instability of the azomethine bond and hence from uncontrollable side reactions of the dialdehyde. The spacer and stable monofunctional aldehyde can be easily joined to agarose by using, e.g., commercially available 4-aminobutyraldehyde diethyl acetal. This compound can be coupled to agarose by the routine cyanogen bromide method via the amino group at alkaline pH where the acetal moiety is stable. The aldehydic function can be released in mild acid hydrolysis. We describe here the coupling of the amino acetal to agarose, acetal hydrolysis and analysis of ethanol formed during the hydrolysis. The resulting derivative of agarose can be used to couple amino ligands to agarose by ordinary methods (1). It is possible for most purposes to bind proteins to this aldehyde gel directly without sodium borohydride reduction incubating the gel with the protein (3). PROCEDURE Fifty milliliters of packed Sepharose 4B 200 (Sigma) are washed with 500 ml of distilled water in a Buchner funnel. The gel is then activated by cyanogen bromide method (4) and is immediately transferred to 25 ml of an ice cold solution of 0.2 M sodium bicarbonate buffer (pH 9.5), containing 4 ml of 4-aminobutyraldehyde diethyl acetal (Merck-Schuchardt). The gel is gently shaken for 16 hr at 4°C and washed with 500 ml of 1 M NaCl and 500 ml of water slowly at room temperature in a funnel to remove the unreacted acetal. The washed acetal-Sepharose gel is then suspended in 100 ml of 100 mM citrate buffer (pH 3.0). The oxygen is removed from the mixture by introducing nitrogen for 5 min after which the vessel is stoppered. The acetal 322 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
SHORT
323
COMMUNICATIONS
is hydrolyzed by gently shaking the suspension for 12 hr at room temperature. The resulting aldehyde-Sepharose is filtered in a Buchner funnel and washed with 100 ml of water. From the filtrate (total volume is measured) ethanol is estimated with a gas chromatograph using 0.05% n-propanol as an internal standard. The glc-analysis of ethanol showed 9 pmol aldehyde groups/ml packed gel (2 mol ethanol equal to 1 mol of aldehyde). The gel is still washed with 500 ml of 0.5 M NaCl and 500 ml of water and stored at 4°C under nitrogen. REFERENCES 1. Parikh, I., March, S. C., and Cuatrecasas, P. (1974) in Methods in Enzymology (Jacoby, W. B., and Wilchek, M., eds.), Vol. 34, Part B, pp. 77-102, Academic Press, London. 2. Weetal, H. H., and Filbert, A. M. (1974) in Methods in Enzymology (Jacoby, W. B., and Wilchek, M., eds.), Vol. 34, Part B, pp. 59-72, Academic Press, London. 3. Kristiansen, T. (1974) in Methods in Enzymology (Jacoby, W. B.. and Wilchek, M.. eds.), Vol. 34, Part B, p. 341, Academic Press, London. 4. March, S. C., Parikh, I., and Cuatrecasas, P. (1974) Anul. Biochem. 60, 149-152. TIMO
KORPELA
ARI HINKKANEN Department of Chemistry and Biochemistry University of Turku SF 20500 Turku 50, Finland Received December I, 1975; accepted December
16, 1975
BIOCHEMISTRY
71, 322-323
(1976)
A Simple Method to Introduce Aldehydic Function to Agarose Agarose has been widely used as a matrix in affinity chromatography and for immobilization of macromolecules. Many chemical methods to prepare these derivatives of agarose have been described (1). Aldehydes react in mild conditions with primary amines to form Schiff bases and thus gels containing aldehyde groups are proper intermediates in immobilizing ligands. Aldehydic function can be well introduced straight into the carbohydrate chains of agarose by sodium metaperiodate oxidation (1). However, this method of synthesis is not convenient when one wants to attach a ligand to the aldehyde group at the end of a spacer. The use of alkylamino support and glutardialdehyde (2) suffers from the instability of the azomethine bond and hence from uncontrollable side reactions of the dialdehyde. The spacer and stable monofunctional aldehyde can be easily joined to agarose by using, e.g., commercially available 4-aminobutyraldehyde diethyl acetal. This compound can be coupled to agarose by the routine cyanogen bromide method via the amino group at alkaline pH where the acetal moiety is stable. The aldehydic function can be released in mild acid hydrolysis. We describe here the coupling of the amino acetal to agarose, acetal hydrolysis and analysis of ethanol formed during the hydrolysis. The resulting derivative of agarose can be used to couple amino ligands to agarose by ordinary methods (1). It is possible for most purposes to bind proteins to this aldehyde gel directly without sodium borohydride reduction incubating the gel with the protein (3). PROCEDURE Fifty milliliters of packed Sepharose 4B 200 (Sigma) are washed with 500 ml of distilled water in a Buchner funnel. The gel is then activated by cyanogen bromide method (4) and is immediately transferred to 25 ml of an ice cold solution of 0.2 M sodium bicarbonate buffer (pH 9.5), containing 4 ml of 4-aminobutyraldehyde diethyl acetal (Merck-Schuchardt). The gel is gently shaken for 16 hr at 4°C and washed with 500 ml of 1 M NaCl and 500 ml of water slowly at room temperature in a funnel to remove the unreacted acetal. The washed acetal-Sepharose gel is then suspended in 100 ml of 100 mM citrate buffer (pH 3.0). The oxygen is removed from the mixture by introducing nitrogen for 5 min after which the vessel is stoppered. The acetal 322 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
SHORT
323
COMMUNICATIONS
is hydrolyzed by gently shaking the suspension for 12 hr at room temperature. The resulting aldehyde-Sepharose is filtered in a Buchner funnel and washed with 100 ml of water. From the filtrate (total volume is measured) ethanol is estimated with a gas chromatograph using 0.05% n-propanol as an internal standard. The glc-analysis of ethanol showed 9 pmol aldehyde groups/ml packed gel (2 mol ethanol equal to 1 mol of aldehyde). The gel is still washed with 500 ml of 0.5 M NaCl and 500 ml of water and stored at 4°C under nitrogen. REFERENCES 1. Parikh, I., March, S. C., and Cuatrecasas, P. (1974) in Methods in Enzymology (Jacoby, W. B., and Wilchek, M., eds.), Vol. 34, Part B, pp. 77-102, Academic Press, London. 2. Weetal, H. H., and Filbert, A. M. (1974) in Methods in Enzymology (Jacoby, W. B., and Wilchek, M., eds.), Vol. 34, Part B, pp. 59-72, Academic Press, London. 3. Kristiansen, T. (1974) in Methods in Enzymology (Jacoby, W. B.. and Wilchek, M.. eds.), Vol. 34, Part B, p. 341, Academic Press, London. 4. March, S. C., Parikh, I., and Cuatrecasas, P. (1974) Anul. Biochem. 60, 149-152. TIMO
KORPELA
ARI HINKKANEN Department of Chemistry and Biochemistry University of Turku SF 20500 Turku 50, Finland Received December I, 1975; accepted December
16, 1975
