The purification of yeast glucose 6-phosphate dehydrogenase by dye-ligand chromatography.
15 Aug 1984-Analytical Biochemistry (Academic Press)-Vol. 141, Iss: 1, pp 79-82
TL;DR: Glucose 6-phosphate dehydrogenase has been purified to homogeneity from baker's yeast by a simple procedure involving affinity elution from a column of red triazine dye, H-8BN, immobilized to Sepharose 6B.
Abstract: Glucose 6-phosphate dehydrogenase (EC 18.104.22.168) has been purified to homogeneity from baker's yeast by a simple procedure involving affinity elution from a column of red triazine dye, H-8BN, immobilized to Sepharose 6B. Eight milligrams of homogeneous protein is obtained in 53% yield from 200 g of dried yeast. This represents the first published purification of the enzyme from Saccharomyces Cerevisiae.
TL;DR: The ability of combining clarification, capture, and purification in a single step will greatly simplify downstream processing flowsheets and reduce the costs of protein purification.
Abstract: The use of expanded beds of STREAMLINE ion exchange adsorbents for the direct extraction of an intracellular enzyme glucose-6-phosphate dehydrogenase (G6PDH) from unclarified yeast cell homogenates has been investigated. It has been demonstrated that such crude feedstocks can be applied to the bed without prior clarification steps. The purification of G6PDH from an unclarified yeast homogenate was chosen as a model system containing the typical features of a direct extraction technique. Optimal conditions for the purification were determined in small scale, packed bed experiments conducted with clarified homogenates. Results from these experiments were used to develop a preparative scale separation of G6PDH in a STREAMLINE 50 EBA apparatus. The use of an on-line rotameter for measuring and controlling the height of the expanded bed when operated in highly turbid feedstocks was demonstrated. STREAMLINE DEAE has been shown to be successful in achieving isolation of G6PDH from an unclarified homogenate with a purification factor of 12 and yield of 98% in a single step process. This ion exchange adsorbent is readily cleaned using simple cleaning-in-place procedures without affecting either adsorption or the bed expansion properties of the adsorbent after many cycles of operation. The ability of combining clarification, capture, and purification in a single step will greatly simplify downstream processing flowsheets and reduce the costs of protein purification. © 1996 John Wiley & Sons, Inc.
TL;DR: Cleaning-in-place (CIP) procedures using 0.5 M NaOH and 4M urea in 60% (v/v) ethanol have demonstrated that the adsorbent can be regenerated with no loss of adsorption capacity of alteration of bed expansion characteristics after many cycles of operation.
Abstract: The use of an expanded bed of STREAMLINE Red H-7B for the purification of the intracellular glycolytic enzyme glucose 6-phosphate dehydrogenase (G6PDH) directly from untreated preparations of disrupted yeast cells has been investigated. Small-scale experiments, carried out in packed beds, have shown that the optimal pH for adsorption is 6.0 and have enabled optimization of elution conditions using a series of eluents. The dynamic capacity of the adsorbent for G6PDH was determined in a small expanded bed to be 28 units/mL. These results were used to develop a preparative scale separation of G6PDH in a STREAMLINE 50 expanded bed column. G6PDH was purified directly from an unclarified yeast homogenate in 99% yield with an average purification factor in the eluted fraction of 103. Cleaning-in-place (CIP) procedures using 0.5 M NaOH and 4M urea in 60% (v/v) ethanol have demonstrated that the adsorbent can be regenerated with no loss of adsorption capacity or alteration of bed expansion characteristics after many cycles of operation.
TL;DR: The case study presented here demonstrates the applicability of general‐purpose membrane adsorbents for the purification of enzymes in recovery of glucose‐6‐phosphate dehydrogenase from yeast.
Abstract: A new adsorbent for the selective binding of enzymes, in the form of microporous membranes carrying triazine dyes as pseudo-affinity ligand, has been implemented in the recovery of glucose-6-phosphate dehydrogenase from yeast. A detailed investigation of the process parameters has been performed. In the adsorption step, the contact time for binding G6PDH could be reduced down to 0.25 s without significant decrease of the capture efficiency. Hence, fast filtration allowed to isolate G6PDH from a dilute extract (1.6 mug G6PDH . mL(-1)), where the enzyme accounted for 1% of the proteins. The yield of the selective elution step using NADP was only 70% at best. It could be improved to near 100% by supplementing the eluent with ethylene glycol, without loss of selectivity. A Scale-up of the cross-section of the membrane by a factor of 40 allowed to purify 1140 U from 0.6 L extract from 1% to 57% purity with 82% yield, within 10 minutes. The case study presented here demonstrates the applicability of general-purpose membrane adsorbents for the purification of enzymes.
TL;DR: With both enzymes, the overall recovery of enzyme activity was greater than 94%, showing that the expanded bed approach to purification was nondenaturing.
Abstract: Malate dehydrogenase (MDH) and glucose 6-phosphate dehydrogenase (G6PDH) have been partially purified from preparations of homogenized yeast cells using Procion Yellow H-E3G and Procion Red H-E7B, respectively, immobilized on solid perfluoropolymer supports in an expanded bed. A series of pilot experiments were carried out in small packed beds using clarified homogenate to determine the optimal elution conditions for both MDH and G6PDH. Selective elution of MDH using NADH was effective but the yields obtained were dependent on the concentration of NADH used. Selective elution was found to be most effective when a low concentration of NaCl (0.1 M) was present. MDH could be recovered in 84% yield with a purification factor of 94 when this strategy was adopted. In the case of G6PDH, specific elution using NADP(+) was successful in purifying G6PDH 178-fold in 96% yield. The dynamic capacity of both affinity supports was estimated by frontal analysis, in an expanded bed with unclarified homogenate, and corresponded to 17 U MDH/mL of settled Procion Yellow H-E3G perfluoropolymer support and 7.7 U H6PDH/mL of settled Procion Red H-E7B perfluoropolymer support. Expanded bed affinity chromatography of MDH resulted in an eluted fraction containing 89% of the applied activity with a purification factor of 113. Expanded bed affinity chromatography of G6PDH resulted in an eluted fraction containing 84% of the applied activity with a purification factor of 172. With both enzymes, the overall recovery of enzyme activity was greater than 94%, showing that the expanded bed approach to purification was nondenaturing.
01 Jan 1966
TL;DR: The results show that the polyacrylamide gel electrophoresis method can be used with great confidence to determine the molecular weights of polypeptide chains for a wide variety of proteins.
Abstract: Forty proteins with polypeptide chains of well characterized molecular weights have been studied by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate following the procedure of Shapiro, Vinuela, and Maizel (Biochem. Biophys. Res. Commun., 28, 815 (1967)). When the electrophoretic mobilities were plotted against the logarithm of the known polypeptide chain molecular weights, a smooth curve was obtained. The results show that the method can be used with great confidence to determine the molecular weights of polypeptide chains for a wide variety of proteins.
TL;DR: It has been shown, in the present paper, that the presence of mercuric chloride in paper strips dyed according to Durrum's technique makes the quantitative elution of protein impossible.
Abstract: 1. 1) It has been shown, in the present paper, that the presence of mercuric chloride in paper strips dyed according to Durrum's technique4 makes the quantitative elution of protein impossible. 2. 2) Bromophenol blue was found not to effect the sensitivity and accuracy of the tannin micromethod2,3. 3. 3) A new technique of fixing protein on paper by tannic acid and simultaneous staining with bromophenol blue (Kado reagent) has been described. 4. 4) The recovery of protein fixed and stained by Kado reagent and eluted with 0.1 N NaOH was found to be complete. 5. 5) A simple and practical method for quantitative estimation of the electrophoretically separated proteins has been described.
TL;DR: Investigation of the effects of solvent ionic strength and pH on the dissociation of the native form A and the proteolytically modified form A′ suggests that these enzymes have a common molecular weight and quaternary structure.
Abstract: A method is described for the purification of two native isoenzymes of yeast hexokinase (A and B) in the presence of phenylmethyl sulphonylfluoride, an inhibitor of yeast proteases. In the absence of this inhibitor two proteolytically modified forms (A′ and B′) were obtained. Investigation of the effects of solvent ionic strength and pH on the dissociation of the native form A and the proteolytically modified form A′ suggests that these enzymes have a common molecular weight and quaternary structure. Both forms exist as dimers of molecular weight 108000 ± 3000 in reversible dissociation equilibrium with monomers of molecular weight 54000 ± 1500. Hexokinase A dissociates to monomer both with increasing ionic strength and in the presence of hexose substrates. Dissociation to constituent polypeptide chains of 54000 molecular weight is effected by maleylation or succinylation of the native enzyme, confirming its dimeric structure. Hexokinase A′ exhibits a pH-dependent dissociation from dimer to monomer. This dissociation is attributable to two ionising groups on the monomer which are predominantly unionised in the dimer.