Affinity Techniques - Enzyme Purification: Part B by Nathan P. Kaplan, Nathan P. Colowick, William B. Jakoby,

By Nathan P. Kaplan, Nathan P. Colowick, William B. Jakoby, Meir Wilchek

The seriously acclaimed laboratory average, Methods in Enzymology, is without doubt one of the such a lot hugely revered guides within the box of biochemistry. in view that 1955, every one quantity has been eagerly awaited, often consulted, and praised through researchers and reviewers alike. The sequence comprises a lot fabric nonetheless suitable this present day - actually a vital booklet for researchers in all fields of lifestyles sciences

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Agarose, particularly in cross-linked bead form, is at present the matrix substance of choice for preparation of stereospecific adsorbents, is The cross-linking of agarose decreases the number of hydroxyl groups and consequently lowers the capacity for introducing ligands. This is of only secondary importance in view of the advantages gained by the increase in alkaline and heat stability and the improvement in several other properties. As will be described later, in some activation procedures crosslinking takes place simultaneously with the introduction of reactive groups for subsequent binding of ligands to the matrix.

Chromatogr. 60, 167 (1971). a°D. A. Rees, Chem. Ind. 16, 630 (1972). 1. 20 COUPLINGREACTIONSAND GENERAL METHODOLOGY [2] Activation step: (~)--OH + X--R--Y *- (~-O--R--Y + HX Coupling step: ( • ) - - O - -+RH2N-Ligand --Y *" (~)--O--R--Y'-Ligand + NHs Activation may also include several steps, as in the case of diazo coupling: (~)--OH + C I C H 2 ~ - N O 2 (~- O-- CH2~-~NO No. < f NH diaz°tizati°n~ ( ~ - O - - C H 2 ~ - N2+ It is important to mention that, after coupling of the desired ligand, reactive Y groups may still be present.

The ideal matrix should meet the following demands: 1. 2. 3. 4. 5. Insolubility Sufficient permeability High rigidity and suitable particle form Zero adsorption capacity Chemical stability under the conditions required for adsorption, desorption, and regeneration 6. Chemical reactivity allowing ligands and spacers to be introduced 7. Resistance toward microbial and enzymatic attack 8. Hydrophilic character No matrix support is ideal in all these respects. Porous glass, for example, has excellent rigidity and permeability but lacks chemical stability in alkaline solution and is indeed not devoid of nonspecific adsorption centers.

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