Christopher R. Lowe

High-Performance Liquid Affinity Chromatography of Proteins on Cibacron Blue F3G-A Bonded Silica

Abstract The reactive triazine dye, Cibacron Blue F3G-A, has been covalently attached to microparticulate porous silica and used for the resolution of a number of complementary proteins by high-performance liquid affinity chromatography (HPLAC). Cibacron Blue F3G-A was converted to its 6-aminohexyl derivative by reaction with 1,6-diaminohexane and coupled to γ-glycidoxypropyltrimethoxysilane-activated porous silica (5 μm) to generate an adsorbent containing 5.5–6.7 μmol dye/g silica. Cibacron Blue F3G-A silica columns, in conjunction with on-line monitoring of protein concentration and enzyme activity, may be exploited for the high speed fully automated resolution of dehydrogenases, isoenzymes, kinases and other proteins such as pancreatic ribonuclease A from simple or complex mixtures. The examples demonstrate the versatility of HPLAC on silica-immobilised Cibacron Blue F3G-A.

High-performance liquid affinity chromatography of enzymes on silica-immobilised triazine dyes

Abstract A number of reactive triazine dyes, including Procion Blue MX-R, Procion Yellow H-A, Procion Green H-4G, Procion Red H-8BN, Procion Brown MX-5BR and Cibacron Blue F3G-A, have been covalently attached to microparticulate silica (10 μm) and used for the resolution of proteins by high-performance liquid affinity chromatography (HPLAC). Purified dyes were either converted to their 6-aminohexyl derivative by reaction with 1,6-diaminohexane and then coupled to γ-glycidoxypropyl-trimethoxysilane-activated silica or coupled directly to glycol-silylated-silica via the reactive triazine ring. These triazine dye-silica adsorbents have been used for the resolution of protein mixtures containing enzymes such as lactate dehydrogenase, hexokinase, alkaline phosphatase, carboxypeptidase G2 and l -tryptophanyl-tRNA synthetase. In addition, the effect of divalent metal ions such as Mg2+ and Zn2+ on promoting the adsorption of metalloenzymes to triazine dye adsorbents has been investigated. These examples illustrate the versatility of triazine dye HPLAC and further demonstrates the speed of analysis, resolution, ease of operation and selectivity of the technique.

Metal ion-promoted binding of proteins to immobilized triazine dye affinity adsorbents

Low concentrations of metal ions, particularly those of the first row transition series such as Zn2+, Co2+, Mn2+, Ni2+, Cu2+, and, to a lesser extent, the group IIA ions, Ca2+ and Mg2+, promotes binding of carboxypeptidase G2, alkaline phosphatase and yeast hexokinase to immobilized Procion Red H-8BN, Procion Yellow H-A and Cibacron Blue F3G-A respectively. The binding of ovalbumin to immobilized Cibacron Blue F3G-A and Procion Orange MX-G is selectively enhanced in the presence of AI3+. With ovalbumin and alkaline phosphatase, the effect is almost totally specific for both the metal ion and dye, whereas with carboxypeptidase G2 and hexokinase, metal ions such as Co2+, Ni2+, Mn2+, Cu2+, Ca2+ and Mg2+ also promote binding to varying degrees. Almost all other monovalent and trivalent metal ions appear to be ineffective. Metal ion-bound enzymes can subsequently be eluted with appropriate chelating agents of the amine, aminocarboxylate or substituted pyridine classes.

Affinity chromatography on immobilised triazine dyes. Studies on the interaction with multinucleotide-dependent enzymes

A systematic investigation into the interaction of several triazinyl dyes with two enzymes from purine metabolism, IMP dehydrogenase (IMP: NAD+ oxidoreductase, EC 1.2.1.14( and adenylosuccinate synthetase (IMP: L-aspartate ligase (GDP-forming), EC 6.3.4.4) has been conducted. Evidence from kinetic inhibition studies, enzyme inactivation with specific affinity labels and specific elution techniques from agarose-immobilised dyes indicate that triazine dyes such as Procion Blue H-B (Cibacron Blue F3G-A), Red HE-3B and Red H-3B are able to differentiate between the nucleotide-binding sites of these enzymes. This information has been exploited to design specific elution techniques for the purification of these enzymes by affinity chromatography.

Preparative high-performance liquid affinity chromatography

The reactive triazine dye, Procion Blue MX-R, has been covalently attached to preparative-grade silica and used for the large-scale purification of rabbit muscle lactate dehydrogenase by high-performance liquid affinity chromatography (HPLAC). Purified dye was coupled directly to glycol-silylated silica via the reactive triazine ring to yield an adsorbent containing 12 mumol dye/g silica. Essentially homogeneous lactate dehydrogenase in 80% overall yield was obtained from crude extracts. Thus this report demonstrates the potential for adapting the speed of operation and resolution shown for triazine dye-HPLAC in analytical applications to preparative protein purification.