Affinity chromatography of bovine heart lactate dehydrogenase using dye ligands linked directly or spacer-mediated to bead cellulose

Abstract

A number of reactive dyes coupled to bead cellulose directly or spacer-mediated has been investigated in respect of their interaction with lactate dehydrogenase (LDH; E.C. 1.1.1.28) from heart muscle. The Procion dyes Red HE-7B and Navy H-ER, as well as the Remazol dyes Brilliant Blue R and Brilliant Red 5-BN directly bound to bead cellulose provide high binding of LDH and the adsorbed enzyme is eluted specifically from these affinity absorbents in high yield. In contrast, under the same conditions no binding of LDH has been found to the Procion dyes Green H-4G, Yellow HE-3G, Scarlet MX-G and Orange MX-G, although an opposite behaviour was expected from the results of affinity partitioning in aqueous two-phase systems. However, immobilizing these dyes via a spacer generated strong binding of the enzyme to the affinity absorbent. The influence of the length of the spacer was studied in respect of the binding capacity and the yield of the enzyme specifically eluted. The applicability of Procion Scarlet MX-G-(diaminohexyl)-bead cellulose for the purification of LDH from muscle extract in one chromatographic step was demonstrated.