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Complications encountered using cibacron blue F3G‐A as a ligand for affinity precipitation of lactate dehydrogenase
Author(s) -
Morris John E.,
Fisher Rod R.
Publication year - 1990
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260360712
Subject(s) - conjugated system , lactate dehydrogenase , chemistry , conjugate , enzyme , nad+ kinase , dehydrogenase , stacking , precipitation , dextran , stereochemistry , biochemistry , organic chemistry , polymer , mathematical analysis , physics , mathematics , meteorology
The use of the affinity interaction between Cibacron Blue F3G‐A (CB) and NADH‐dependent enzymes to selectively precipitate these enzymes has been examined. An attempt was made to form crosslinked precipitates of lactate dehydrogenase (LDH) using bis‐ and poly‐CB conjugates. When precipitation was not observed, an examination of the interaction between the enzyme and the conjugated CB was made. Quasielastic light scattering indicated only a slight radius increase, the greatest being from 50 to 130 Å, when a CB‐dextran conjugate was added to a solution of LDH, and no increase when bis‐CB made with a 1, 6‐diaminohexane spacer was added to a similar solution. The results of enzyme inhibition studies showed that conjugated CB bound at the NAD + site of LDH. Spectral measurements of the conjugated CB below 5 μ M were similar to those reported for a stacking interaction that occurs in solutions with CB concentrations above 5 μ M We conclude that the conjugated CB is binding to the LDH, but that a competing dye stacking interaction prevents extensive cross‐linking of the LDH, and thus inhibits precipitation.