Hydrophobic interaction column

Protein extraction procedures employing chemicals such as detergents are effective in many instances, but they suffer from a number of drawbacks, not least of which is that they often induce protein denaturation and precipitation. This obviously limits their usefulness. Furthermore, even if the chemicals employed do not adversely affect the protein, their presence may adversely affect a subsequent purification step (e.g. the presence of detergent can prevent proteins from binding to a hydrophobic interaction column). In addition, the presence of such materials in the final preparation, even in trace quantities, may be unacceptable for medical reasons. 

Growth conditions of Synechocystis sp. PCC 6803 and subsequent isolation of thylakoid membranes were described in literature [8], From these membranes, PS I was extracted by dodecyl-B-D-maltoside treatment and purified by two successive HPLC steps, using first an anion exchange column (Poros 50 HQ, PerSeptive) and second a hydrophobic interaction column (Poros 20 Butyl, PerSeptive). The purification of trimeric PS I is described in detail in literature [9],... 

Because HIC is based on surface-tension phenomena, changing those characteristics by the addition of surfactants affects retention. In a study of the effects of surfactants on retention of proteins by HIC, the addition of CHAPS 3-[(3-cholamidopropyl) di-methylammonio]-l-propane sulfonate to the mobile phase resulted in shortened retention, improvement of peak shape, and a change in peak order for enolase and bovine pancreatic trypsin inhibitor [8]. The effects were dependent on the concentration of the surfactant. Surfactants can usually be washed easily from hydrophobic interaction columns because the bonded phases are neither highly hydrophobic nor ionic. 

In addition, a PHA depolymerase fi-om P. stutzeri (strain YM1006) also prepared by a hydrophobic interaction column chromatography. The enzyme firom P. stutzeri showed a single band on the gel of SDS-... 

Occasionally a second ferredoxin which elutes at lower ionic strength (0.2 - 0.3 M NaCl) is observed and this is called ferredoxin II. We found that ferredoxin II was ten times as active as ferredoxin I. Hutson et al. (5) had found that these ferredoxins differed by a factor of two in supporting the reduction of NADP by Photosystem I and differed less in the decarboxylation of pyruvate by the phosphoroclastic system of Clostridium pasteruanum. We applied the ferredoxin II fraction from the ion exchange column to a hydrophobic interaction column and resolved it into three distinct isozymes. One of these was very active in the cytochrome reduction activity, the second less active and the third isoz3nne was inactive. This specificity of interaction of one ferredoxin with low potential... 

Hydrophobic interaction columns separate proteins using hydrophobicity, similar to that of reversed-phase HPLC. However, this method provides an alternative approach with little or no denaturation of proteins because organic solvents are not required for the elution. In fact, ceruloplasmin, which was denatured during reversed-phase chromatography could be eluted from a hydrophobic interaction column without denaturation (Fig. 6). /f2-Glycoprotcin I, which has been... 

When a protein is adsorbed to a hydrophobic surface, denaturation is often not an instantaneous phenomenon. Thus, the extent of unfolding will increase with increasing time of adsorption. Both Karger and Hearn et al. [40] have monitored this residency effect on reversed-phase and hydrophobic interaction columns using a combination of techniques including derivative spectroscopy. The Hearn group also studied denaturation kinetics in size exclusion chromatography in the presence of the powerful denaturant urea. 

Mendieta-Taboada, O., E. S. Kamimura, and R Maugeri. 2001. Modelling and Simulation of the Adsorption of the Lipase from Geotrichum Sp. on Hydrophobic Interaction Columns. Biotechnology Letters 23 (10) 781-786. 

FIGURE l.l Hydrophobic interaction and reversed-phase chromatography (HIC-RPC). Two-dimensional separation of proteins and alkylbenzenes in consecutive HIC and RPC modes. Column 100 X 8 mm i.d. HIC mobile phase, gradient decreasing from 1.7 to 0 mol/liter ammonium sulfate in 0.02 mol/liter phosphate buffer solution (pH 7) in 15 min. RPC mobile phase, 0.02 mol/liter phosphate buffer solution (pH 7) acetonitrile (65 35 vol/vol) flow rate, I ml/min UV detection 254 nm. Peaks (I) cytochrome c, (2) ribonuclease A, (3) conalbumin, (4) lysozyme, (5) soybean trypsin inhibitor, (6) benzene, (7) toluene, (8) ethylbenzene, (9) propylbenzene, (10) butylbenzene, and (II) amylbenzene. [Reprinted from J. M. J. Frechet (1996). Pore-size specific modification as an approach to a separation media for single-column, two-dimensional HPLC, Am. Lab. 28, 18, p. 31. Copyright 1996 by International Scientific Communications, Inc.. Shelton, CT.]... 

Electrostatic effects have long been recognized in commercial HPLC columns for SEC of proteins (15,21,22). The usual remedy is to add 100 mM salt to the mobile phase. This works here too the Lys and Asp peaks collapse into the Gly peak with 100 mM salt (Eig. 8.8). High concentrations of sodium sulfate were added to determine the role played in SEC by hydrophobic interactions (sodium sulfate, a structure-forming salt, strengthens such interactions). Sodium sulfate increased the retention only of the most hydrophobic amino acids to any extent, and then only when the concentration approached 1 M. Clearly, hydrophobic interaction cannot account for the elution order of amino acids on PolyHEA. 

The protein can be further purified by hydrophobic interaction chromatography on a column of Butyl Sepharose 4 Fast Flow (Pharmacia elution with decreasing concentration of (NH4)2S04 starting at 1.5 M), and gel filtration on a column of Superdex 200 Prep (Pharmacia Inouye et al., 2000). 

Step 2. Hydrophobic interaction chromatography on a column of Phenyl-Sepharose CL-4B. The sample was adsorbed on the column in the basic buffer containing 0.5 M (NH SC. The photoprotein adsorbed was first washed with the same buffer, then eluted with the basic buffer. 

Fig. 8. Preparative isolation of hexon antigen of EDS-76 by hydrophobic-interaction chromatography on Butyl-PG column (2x5 cm) (A) application of the allantoic fluid diluted (1 5) by 50 mM potassium acetate, pH 4,130 ml (B)0.01 mol/1 potassium acetate, pH 5.5 (C) 0.01 mol/1 potassium bicarbonate pH 8.0, 10% isopropanol (D) 0.01 mol/1 potassium carbonate pH 9.6, 10% isopropanol. EDS-0 — components of alantoic fluid eluted with buffer A, EDS-1 — desorbed hexon fraction eluted with buffer C, EDS-2 — fraction desorbed with buffer D [56]...

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