Two Elution Mechanisms of MEP Chromatography

2018 ◽  
Vol 20 (1) ◽  
pp. 28-33 ◽  
Author(s):  
Tsutomu Arakawa ◽  
Masao Tokunaga ◽  
Takuya Maruyama ◽  
Kentaro Shiraki
Keyword(s):  
Hydrophobic Interaction ◽  
Positive Charge ◽  
Hydrophobic Interaction Chromatography ◽  
Normal Operation ◽  
Acidic Ph ◽  
Charge Induction ◽  
Pyridine Group ◽  
Ph Range ◽  
Hydrophobic Charge Induction Chromatography ◽  
Positively Charged

MEP (mercapto-ethyl-pyridine) HyperCel is one of the hydrophobic charge induction chromatography (HCIC) resins. Under normal operation, proteins are bound to the MEP resin at neutral pH, at which MEP is not charged, mostly via hydrophobic interaction. MEP has a pyridine group, whose pK is 4.8, and hence is positively charged at acidic pH range. Based on the binding mechanism (i.e., hydrophobic interaction) and the induced positive charge at acidic pH, there may be two ways to elute the bound proteins. One way is to bring the pH down to protonate both MEP resin and the bound protein, leading to charge repulsion and thereby elution. Another way is to use hydrophobic interaction modifiers, which are often used in hydrophobic interaction chromatography, to reduce hydrophobic interaction. Here, we summarize such two possible elution approaches.

scholarly journals Influence of particle properties on iron flocculation

2017 ◽  
Vol 18 (5) ◽  
pp. 1617-1624
Author(s):  
D. J. de Ridder ◽  
D. van Halem
Keyword(s):  
Positive Charge ◽  
Water Phase ◽  
Significant Delay ◽  
Iron Species ◽  
Particle Properties ◽  
Charged Species ◽  
Ph Values ◽  
Ph Range ◽  
Positively Charged ◽  
Charge Interactions

Abstract In this study, the importance of charge interactions during flocculation of Fe3+ in the presence of particles and anions/cations at various pH values was investigated. SiO2, (s) and ZnO(s) were dosed as particles to promote charge interactions and/or serve as a nucleus to accelerate floc formation. In the pH range 6–9, SiO2, (s) is negatively charged, while ZnO(s) carries a positive charge. Ca2+ and HPO42− were selected to investigate charge interactions in the water phase. A significant delay in floc growth due to charge repulsion between negatively charged iron species was observed at pHini 9. For positively charged species at pHini 6, a delay in floc growth was observed as well, but to a lesser degree. These effects could be neutralized by either dosing (positively charged) ZnO(s) or Ca2+ at pHini 9, or (negatively charged) SiO2, (s) at pHini 6. The addition of phosphate did not hinder floc growth at pHini 6. While phosphate completely inhibited floc growth at pHini 7–9 in the presence of negatively charged SiO2, (s), the presence of positively charged ZnO(s) partly neutralized the detrimental influence of phosphate on floc growth. Similarly, dosing Ca2+ partly neutralized the effect of phosphate.

Purification of Glycerol Kinase by "Dye-Ligand" Chromatography and Hydrophobic Interaction Chromatography on Bead-Cellulose Derivatives

1993 ◽  
Vol 58 (2) ◽  
pp. 445-451 ◽  
Author(s):  
Vladimír Žúbor ◽  
Albert Breier ◽  
Marta Horváthová ◽  
Dagmar Hagarová ◽  
Peter Gemeiner ◽  
...  
Keyword(s):  
Hydrophobic Interaction ◽  
Specific Activity ◽  
Hydrophobic Interaction Chromatography ◽  
Glycerol Kinase ◽  
Enzymic Activity ◽  
Cellulose Derivatives ◽  
Long Term Storage ◽  
Bead Cellulose ◽  
Term Storage

The crude extract of cytosole enzymes was obtained from homogenized cells of Saccharomyces cerevisiae by partition. The enzyme was then isolated from the lower aqueous phase displaying higher glycerol kinase activity by dye-ligand chromatography on Cibacron Blue (CB) or Remazol Brilliant Blue R (RB)-derivatized bead-cellulose, ATP being the eluent. The specific activity of glycerol kinase rised more than 10 and 7-times after affinity dye-ligand chromatography and hydrophobic interaction chromatography, respectively. Glycerol kinase obtained by the latter method was purified by CB-bead cellulose. The final preparation maintained its enzymic activity without noticeable losses during a long-term storage at 4 °C in dark.

Positive charge modifications alter the ability of XIP to inhibit the plasma membrane calcium pump

1996 ◽  
Vol 271 (3) ◽  
pp. C736-C741 ◽  
Author(s):  
W. Xu ◽  
C. Gatto ◽  
M. A. Milanick
Keyword(s):  
Plasma Membrane ◽  
Positive Charge ◽  
Calcium Pump ◽  
Inhibitory Peptide ◽  
Ca Pump ◽  
Contact Points ◽  
Plasma Membrane Calcium Pump ◽  
Peptide Analogues ◽  
Positively Charged

Exchange inhibitory peptide (XIP; RRLLFYKYVYKRYRAGKQRG) is the shortest peptide that inhibits the plasma membrane Ca pump at high Ca (A. Enyedi, T. Vorherr, P. James, D. J. McCormick, A. G. Filoteo, E. Carafoli, and J. T. Penniston, J. Biol. Chem. 264: 12313-12321, 1989). Sulfosuccinimidyl acetate (SNA)-modified XIP does not inhibit the Ca pump; SNA neutralizes the positive charge on Lys at positions 7, 11, and 17. Peptide 2CK-XIP (RRLLFYRYVYRCYCAGRQKG) inhibits the pump, but the iodoacetamido-modified peptide does not inhibit. Three peptide analogues, in which 7, 11, and 17 were Ala, Cys, or Lys, inhibited about as well as XIP. SNA modification of these analogues (each with 1 Lys) did not inhibit. SNA modification of 2CK-XIP results in a peptide that does not inhibit; thus position 19 is important. Our results suggest that it is critical that position 19 be positively charged, that positions 7, 11, and 17 are important contact points between XIP and the Ca pump (with at least one positively charged), and that, whereas it is not essential that residues 12 and 14 be positive, they cannot be negative.

Hydrophobic interaction chromatography of proteins

2007 ◽  
Vol 1141 (2) ◽  
pp. 235-243 ◽  
Author(s):  
Brian C.S. To ◽  
Abraham M. Lenhoff
Keyword(s):  
Hydrophobic Interaction ◽  
Hydrophobic Interaction Chromatography

Poly(β-L-malate) hydrolase from Plasmodia of Physarum polycephalum

1995 ◽  
Vol 41 (13) ◽  
pp. 192-199 ◽  
Author(s):  
Christian Korherr ◽  
Michael Roth ◽  
Eggehard Holler
Keyword(s):  
Hydrophobic Interaction ◽  
Malic Acid ◽  
Physarum Polycephalum ◽  
Culture Medium ◽  
Specific Activity ◽  
Hydrophobic Interaction Chromatography ◽  
Reduced Form ◽  
Serine Esterase ◽  
Hydroxybutyric Acid ◽  
Ph Optimum

A 68-kDa extracellular glycoprotein from Physarum polycephalum that hydrolyses specifically poly(β-L-malic acid) by removing monomers of L-malic acid in an exolytic manner has been purified and characterized. The enzyme was purified 1740-fold from the culture medium by ammonium sulfate precipitation, hydrophobic interaction chromatography on butyl-Toyopearl, and gel permeation chromatography on Superdex 200 to a specific activity of 9.0 μmol∙min−1∙mg−1. The hydrolase was also purified from the cytosol, which contained 1 mg in 43 g cells in contrast to 1 mg extracellular enzyme in 28 L of culture medium. The pH optimum was pH 3.5 as a result of the effect of an acidic side chain on Vmax and the preferred binding of poly(β-L-malate) in the ionized form. Intracellular hydrolase was only marginally active on [14C]poly(β-L-malate) that had been injected into plasmodia. Poly(L-aspartate), poly(L-glutamate), poly(vinyl sulfate), and poly(acrylate) were neither bound nor degraded by the hydrolase. Poly(β-hydroxybutyric acid), which was considered the reduced form of poly(β-L-malate), was not a substrate. The enzyme is neither a metallo- nor a serine-esterase, and is distinct from poly(3-hydroxybutyric acid) depolymerases. It is related to a glucosidase with respect to hydrophobic interaction chromatography, the pH-activity dependence, and its inhibition with mercuribenzoate, N-bromosuccinimide, and D-gluconolactone, but not the use of the substrates.Key words: poly(β-L-malate), polymalatase, Physarum polycephalum, biodegradative polymer.

scholarly journals Fractionation and recovery of whey proteins by hydrophobic interaction chromatography

2011 ◽  
Vol 879 (7-8) ◽  
pp. 475-479 ◽  
Author(s):  
Maria João Santos ◽  
José A. Teixeira ◽  
Lígia R. Rodrigues
Keyword(s):  
Hydrophobic Interaction ◽  
Whey Proteins ◽  
Hydrophobic Interaction Chromatography
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