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Additional info for Affinity Techniques - Enzyme Purification: Part B
The carboxyl group density may be measured by titration as described under Analytical Procedures. Preparation of Hydrazide Derivatives (H). The dry beads are added gradually with stirring to an aqueous solution of hydrazine (1 M to 6 M ) . lr The suspension is stirred slowly or swirled occasionally while being held at a constant temperature. The reaction is stopped after the desired time by funnel washing of the gel. 2 M NaC1 are used if flow rates permit. Washing is completed by a series of batch treatfnents until supernatants (free of beads) become negative or show only pale violet color in the TNBS test.
Porath, Biochim. Biophys. Acta 184, 93 (1969). 33G. I. -U. Fisch, and R. Schwyzer, FEBS Lett. 13, 56 (1972). 34j. Porath and L. Sundberg, Protides Biol. Fluids, Proc. Colloq. 18, 401 (1970). L. Sundberg and J. Porath. To be published.  GENERAL METHODS AND COUPLING PROCEDURES 25 hydroxyl in the polymer matrix, leaving the other group free ; and (2) the coupling wherein the oxirane ether is allowed to react with the ligand-forming substance. 35 The following procedures are recommended as exemplified by 6% agarose gel (Sepharose 6B) .
They will thus couple to amines, alcohols, and phenols at lower temperatures and at lower pH than the oxirane intermediates described in the preceding section . The divinylsulfone coupling method is well suited for the attachment of hydroxylic compounds, R-OH, to form products presumably containing the following connector-spacer structure : M-O-CH2 CH2 SO 2-CH2 -CH2 -OR Unfortunately, the products are unstable in alkaline solution (the amino link above about pH 8 and the hydroxyl link at about pH 9 or 10) .
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