Gel filtration study of mushroom proteins

Summary Proteins from six edible wild mushrooms were extracted by sodium chloride, ethanol or sodium hydroxide and analysed by gel filtration on Sephacryl S-200. Analysis of the whole extracts and the separated proteins of different molecular weight as well as the determination of their amino acid content demonstrated that the soluble mushroom proteins are dimers, trimers and decamers of the simple main protein (polypeptide) with an approximate molecular weight of 15,000. The polymers were stabilized by both covalent and non-covalent forces as demonstrated by gel filtration in acetic acid before and after reduction of the covalent bonds.     

Chromatographic behaviour of aromatic compounds on gel filtration Media

Summary Highly cross-linked gels utilized as gel filtration media exhibit adsorption effects towards aromatic or apolar compounds. The adsorption is linearly dependent on salt concentration. We investigated the effect of various salts spanning the Hofmeister series on the adsorption of three model compounds, Adenosine 5′-phosphate, ε-dinitrophenyllysine and tryptophan. It was found that salts exhibiting marked salting-out properties, such as potassium phosphate and sodium sulfate, were the most effective in enhancing the adsorption were the most effective in enhancing the adsorption of the aromatic molecules onto the gel. On the contrary, in the presence of salts with marked salting-in properties, such as Kl or KSCN, the linear dependence of elution parameters for the same compounds was negatively correlated and adsorption decreased with salt concentration. However, a number of salts of intermediate character in the Hofmeister series, such as NaCl or NH₄Cl, did not affect the elution behaviour of the chosen model compounds. The different effects observed are probably due to differential hydration of the molecules in the presence of the salts and to the consequent different hydrodynamic behaviour of the solute when interacting with the hydration layers of the gel matrix.     

Endcapping of high performance liquid chromatography packings derived from silica gel

The effect of endcapping a reversed phase chromatographic packing material derived from silica gel depends on the degree of derivatization of the phase. The effect can be dramatic for phases with low loading. Whether an octadecylated silica gel has been endcapped or not can be ascertained by chromatographing the pair naphthalene/1-nitronaphthalene with a water/methanol eluent producing at least k' ≈ 10 for naphthalene. The ratio of the two k' values is 1.4 or higher for an endcapped material, while it is only 1.1 -1.2 for a non-endcapped phase. A similar approach fails to give conclusions of similar utility for octylated silica gels.     

Separation studies of amino acids,proteins and enzymes on bonded 1,2-dihydroxy-, 1,2-hydroxylamino-and amino silica packings

Summary 1,2-dihydroxy-3-propoxypropyl (HPPS), 1-amino-2-hydroxy-3-propoxypropyl (AHPS) and 1-aminoethyl-3-aminopropyl (AEAPS) silica were synthesized by means of both a surface modification procedure (I) and a bulk modification procedure (II). Method (I) gave a surface concentration, , of functional groups of 2–3 mole/m², whereas method (II) gave values up to 5 mole/m². Retention times, peak asymmetries and plate heights of thiamine and ascorbic acid eluted with aqueous buffer solutions ranging from pH 5.3 to 9.2 gave only a±5% variation over periods of 12 hours and more.The recoveries of selected enzymes and proteines examined under static and dynamic conditions were between 60% and 100% depending on the functional group of the packing and on the solute. With HPPS silica, which showed the least adsorption and denaturing effects, separation of biopolymers depended on a size exclusion effect in the relative molecular mass range 10,000 to 80,000 and could be achieved within 10 min.A part of the paper was presented at the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Febr. 28 to March 4, 1977, Cleveland, Ohio, U.S.A.     

Influence of nonionic surfactants on the chromatographic behaviour of proteins in hydrophobic interaction chromatography

The chromatographic characteristics of proteins in the presence of additives of nonionic surfactants Brij-35 and Tween-80 in the conditions of descending gradient of ammonium sulfate and phenyl-coated polymeric stationary phase were investigated. It was revealed that retention factors of proteins may be regulated by use of mentioned additives. The improvement of resolution is achieved for some hardly separated pairs of proteins, viz. albumin egg/albumin bovine, aldolase/tripsin. A reversion of the elution order is observed for tripsin/chymotrypsinogen A.     

Specific salt effects in hydrophobic interaction chromatography of proteins

Summary It has been noted in the literature that certain salts enter into specific interaction with proteins. As a result of this, they may act as salting-in agents. We have investigated the effect of magnesium chloride which is known to possess such unusual properties on the retention of proteins in hydrophobic-interaction chromatography. First the retention behaviour of amino acids and small peptides having a wide polarity range was studied on reversed-phase columns using eluents containing (NH₄)₂SO₄, MgSO₄ or MgCl₂, in wide the concentration ranges. For less polar eluites plots of the logarithmic retention factors against the salt concentration were found to be linear, whereas the more polar species showed irregular behavior. The retention of a wide range of proteins was measured on a TSK Phenyl-5-PW column using eluents containing (NH₄)₂SO₄, MgSO₄ or MgCl₂ at different concentrations.The salt-mediated retention was regular with (NH₄)₂ SO₄ and MgSO₄ although MgSO₄ showed a lesser effect than that predicted by the surface-tension increment. The effect of MgCl₂ was quite irregular: the retention factors either increased or decreased or remained unchanged depending on the protein. These results corroborate earlier observations regarding the particular effect of MgCl₂ and suggest the modulation of selectivity in hyrophobic-interaction chromatography by the addition of MgCl₂ to the eluent.