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Rapid screening of dyes employed as affinity ligands to purify enzymes from yeast - I

This paper describes a rapid method for screening potential dye ligands for use in affinity chromatography.

Textile dyes were non-covalently coupled to a cross-linked polysaccharide Sepharose matrix. Yeast alcohol dehydrogenase (ADH) was used as the model protein for evaluating the screening system. A homogenate from baker’s yeast was used as the crude source of enzyme. Batchwise adsorption and elution were used to evaluate the individual dyes. The influence of pH and ionic strength in the binding and elution steps was evaluated.

Batch isotherms were used to evaluate parameter characteristics. Experimental data obtained were fitted to Langmuir isotherms to determine the maximum binding capacity and the dissociation constant for each dye evaluated in this study. A dynamic binding capacity of 107.6 units of ADH/g of resin was determined for Procion Turquoise MXG dye by frontal analysis.

Specific elution with NAD and non-specific elution with 50 mM Tris/HCl buffer, pH 8.5, were tested when Procion Turquoise MXG was used, giving purification factors of 53.5 and 4.4 respectively. This screening technique is inexpensive and can be performed in a few hours. It was possible to predict the performance of different reactive dyes in this way, and the influence of pH and salt on the binding behaviour was demonstrated.

Introduction

Downstream processing is considered a critical step in the commercial development of biotechnology. Of the various techniques, affinity chromatography remains the most powerful. Some of the ligands used in enzyme purification have been reactive triazine dyes. They are stable, easy to immobilise, inexpensive, readily available and have a high binding capacity.

Reactive dyes are also employed in different affinity purification techniques such as affinity precipitation, aqueous two-phase partitioning, expanded-bed chromatography and dye bound on to membrane for affinity purification. The introduction of dye-ligand adsorbents for protein isolation some years ago added an additional mode of adsorption to those available at the time. The reactive dye Cibacron Blue F3GA has been used especially in the purification of proteins. Many other dyes can interact with proteins and other biomolecules and are therefore of potential use in affinity chromatography.

There has been a reluctance to use textile dyes on a large scale for therapeutically applicable proteins for fear of possible dye leakage and consequent contamination. A series of toxicity investigations in vitro with eukaryotic cells and with prokaryotic cells for genotoxic studies demonstrated zero or slight toxicity for Reactive Blue 2 and Reactive Red 120. Several strategies for the screening of dye-ligands have been proposed. Taking into account the binding capacity of alcohol dehydrogenase (ADH), we have screened a large number of dyes by adsorption isotherms. This screening technique is inexpensive and can be performed in a few hours. It was possible to predict the performances of different reactive dyes in this way. The influences of pH and salt on the binding behaviour were demonstrated. Affinity chromatographies for ADH purification were performed with specific and non-specific elution by using Procion Turquoise MXG.

Materials and methods Chemicals

NAD+, sodium pyrophosphate, sodium phosphate (monobasic and dibasic), Trizma and Cibacron Blue F3GA were purchased from Sigma (St. Louis, MO, U.S.A.). Commercial baker’s yeast from Duquesa S.A. (Tucuma! n, Argentina), packed as moist 500 g cakes, was purchased from a local store. Cibacron

Cibacron Red 3BA, Cibacron Turquoise 6GE and Cibacron Turquoise PGF (industrial grade) were gifts from Ciba Geigy (Buenos Aires, Argentina). Procion Yellow HE4R, Procion Abbreviation used: ADH, alcohol dehydrogenase. Orange HER, Procion Blue HEGN 125 per cent, Procion Blue MXG, Procion Blue HERD, Procion Marine HER 150, Procion Red HE7B, Procion Red HE3B, Procion Ruby MXB, Procion Turquoise MXG, Procion Turquoise HA and Procion Green HE4BD were gifts from ICI (Buenos Aires, Argentina). Vilmax Blue 2R, Vilmax Blue 5R and Vilmax Red 5B were gifts from Vilmax (Buenos Aires, Argentina). All other chemicals were of analytical grade.

Preparation of yeast cream

An alcoholic fermentation was performed before preparation of the baker’s yeast (Saccharomyces cerevisiae) homogenate. The fermentation medium contained (in g/l) : sucrose 200, (NH4)2SO4 4, potassium phosphate 0.5. An 8- litre fermenter was inoculated with 350 g of commercial baker’s yeast (30 per cent dry weight). The anaerobic process continued for 16 h at 30øC. Cells were harvested by centrifugation at 16000 g for 5 min at 4øC. The yeast cream (20 per cent dry weight) was stored at 20øC.

Measurement of protein content

Total protein concentration was determined by the Coomassie Blue binding assay. BSA was used as the standard protein. Absorbance was measured at 595 nm.

Determination of ADH activity

Enzymic activity was measured in accordance with the Worthington Enzyme Manual.

Preparation of yeast homogenate

Wet baker’s yeast (20 g, 20 per cent dry weight) was treated with 50 ml of 0.25 M disodium phosphate. The suspension was incubated at 37øC for 2.5 h to disrupt the cells. The supernatant was precipitated with (NH4)2SO4 to 60 per cent satn. on a rocking table at room temperature (24-26øC) for 30 min. It was centrifuged at 5000 g for 5 min and the pellet was resuspended in 50 mM phosphate buffer, pH 6.4. The resuspended solution was dialysed against 50 mM Tris}HCl, pH 7.0, at 4øC in dialysis tubing with a molecular mass cutoff of 12000 Da.

Immobilisation of dye on support

Distilled water (50 ml) was mixed with 1.5 g of dye and 75 g of Sepharose CL4B. After 10 min at room temperature, 3.0 g of disodium carbonate (final pH 10.8) was added and mixed for 48 h at 45øC. The dyed supports were then washed sequentially on glass filters with NaCl (1 M, 500 ml), citrate buffer (50 mM, pH 3.0, 500 ml) and Tris/HCl buffer (50 mM, pH 9.0, 500 ml), then stored in 50 mM sodium phosphate buffer, pH 6.4.

Dye screening by adsorption isotherms

Eppendorff tubes containing 0.1 g of dye - Sepharose received 1 ml of yeast homogenate (28 units/ml ADH; 8 mg/ml protein) diluted to different concentrations (10 per cent, 20 per cent, 50 per cent and 100 per cent) with 50 mM sodium phosphate buffer, pH 6.4. After 15 min, samples were taken and ADH activity and protein content were determined. The amount of enzyme bound to the adsorbent (q*) was calculated as the total amount of enzyme present at the beginning of the experiment minus the amount remaining in the soluble phase at equilibrium (c*).

(The second part of this paper will be published in the issue dated November 21, 2002).