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By Alton Meister

Advances in Enzymology and similar parts of Molecular Biology is a seminal sequence within the box of biochemistry, providing researchers entry to authoritative experiences of the most recent discoveries in all components of enzymology and molecular biology. those landmark volumes date again to 1941, offering an unmatched view of the historic improvement of enzymology. The sequence deals researchers the newest knowing of enzymes, their mechanisms, reactions and evolution, roles in complicated organic method, and their software in either the laboratory and undefined. every one quantity within the sequence good points contributions by way of prime pioneers and investigators within the box from around the globe. All articles are rigorously edited to make sure thoroughness, caliber, and clarity.

With its wide variety of themes and lengthy historic pedigree, Advances in Enzymology and similar components of Molecular Biology can be utilized not just by means of scholars and researchers in molecular biology, biochemistry, and enzymology, but in addition by means of any scientist attracted to the invention of an enzyme, its homes, and its purposes.

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5. Gcfter, M. , and Russell, R. , J . Mol. , 39, 145 (1969). 6. , and Zachau, H. , Eur. J . , 5, 546 (1968). 7. , and Hall, R. , Biochem. Biophys. Res. , 25, 441 (1966). 8. Capra, J. , J . Mol. ,33, 591 (1968). 9. , Cold Spring Harbor S y m p . Qunnt. , 28, 139 (1963). 10. Agarwal, K. , Caruthers, M. , Khorana, H. , Rajbhandary, U. , Van de Sande, J. , Nature, 227, 27 (1970). 11. Bumstead, R. , Dahl, J. , and Strominger, J. , J. Biol. , 243, 770 (1968). 12. Stewart, T. , Roberts, R. , and Strominger, J.

Agarose a. General Considerations b. Specific Procedures c. Stability of Derivatives 3. Acrylamide D. Linking of Proteins to Agarose V. Adsorption and Elution of Affinity Columns VI. Selected Applications A. Purification of Enzymes 1 . Staphylococcal Nuclease 2. B-Galactosidase 3. Proteolytic Enzymes 4. Neuraminidases 5. Acetylcholinesterase 6. Tyrosine Aminotransferase 7. Avidin 8. CAMP-dependent Protein Kinases 9. Chorismate Mutase 10. T 4 Bacteriophage-specific Dihydrofolatc Reductase 11. Miscellaneous Enzymes B.

The resulting yellow protein was applied to an affinity column; the enzyme adsorbed as a very sharp, intense yellow band at the top of the column. 1 M acetic acid caused a rapid downward movement of the sharply demarcated yellow band. The above considerations also explain why one of the most effective means of eluting a strongly adsorbed enzyme is to remove the uppermost portion of the adsorbent and t o dilute this in a beaker with a relatively large volume of an appropriate buffer. 111. Selection of Insoluble Carriers An ideal insoluble carrier for affinity chromatography of enzymes should possess the following properties: i t must interact very weakly with proteins in general t o minimize the nonspecific adsorption of proteins; it should exhibit good flow properties that are retained after coupling; i t must possess chemical groups that can be activated or modified under conditions innocuous t o the structure of the matrix t o allow the chemical linkage of a variety of ligands (these chemical groups should be abundant in order t o allow attainment of a high effective concentration of coupled inhibitor, that is, capacity, so that satisfactory adsorption can be obtained even with protein-inhibitor systems of low affinity); i t must be mechanically and chemically 38 PEDRO CUATRECASAS stable to the conditions of coupling and t o the various conditions of pH, ionic strength, temperature, and presence of denaturants, such as urea, guanidine-hydrochloride,and detergents which may be needed for adsorption or elution because such properties also permit repeated use of the specific adsorbent; and it should form a very loose, porous network that permits uniform and unimpaired entry and exit of large macromolecules throughout the entire matrix.

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