Gel Filtration Chromatography Of Proteins Research Articles

Characteristics of Superdex® prep grade media for gel filtration chromatography of proteins and peptides

Some chemical and chromatographic properties of Superdex® 30 prep grade, Superdex 75 prep grade and Superdex 200 prep grade, all new composite media for separation of peptides and proteins, were investigated. The selectivity of Superdex 30 prep grade was studied by constructing calibration graphs ( K av vs log M r) of peptides and poly(ethylene glycol) (PEG) standards. The influence of the sodium chloride concentration of the mobile phase on the distribution coefficient of peptides demonstrates that Superdex 30 prep grade contains small amounts of charged groups. Titration results show that the amount of negatively charged groups (carboxyl groups) was about 1 μmol/ml gel for all three investigated Superdex media. A homologous series of n-alphatic (n=4–8) alcohols was used to study the hydrophobic interaction behaviour of Superdex media. The effects of temperature, salt concentration and methanol on K av of the alcohols were used to characterize the elution mechanism. Hydrophobic interactions increased with the amount of dextran coupled to the agarose bead. This means that of the three Superdex media, Superdex 200 prep grade is the least hydrophobic. The functional stability of Superdex 30 prep grade was studied by testing the separation of PEG standards and peptides during repeated cleaning-in-place (CIP) treatments with 1·0 m sodium hydroxide solution and 0·1 m hydrochloric acid. The separation behaviour of the PEG standards and the peptides was unaltered after the medium had been treated for a total contact time of 400 h in 0·1 m hydrochloric acid. However, a slight increase in retention volume was observed after treatment in basic conditions. This effect is a result of hydrolysis of covalently bonded dextran in the agarose pores. In basic conditions, the highest carbohydrate (dextran) leakage was observed for Superdex 30 prep grade and the lowest for Superdex 200 prep grade. In acidic conditions it was found that for all Superdex media, dextran and low amounts of 5-(hydroxymethyl)-2-furaldehyde (5-HMF) were released but the separation characteristics were unaltered after treatment in 0·1 m hydrochloric acid. The highest 5-HMF leakage was observed for Sepharose High Performance (the agarose bead on which Superdex media are based). The lowest amount was released from Superdex 30 prep grade.

Glucose-silica, an improved medium for high-pressure gel filtration chromatography

Glucose was covalently coupled to aminopropyl derivatized silica of various pore sizes. The reaction most likely involved Schiff's base formation between the aldehyde of glucose and the amine on the silica which was then reduced with NaCNBH3. This glucose-silica behaved as a nearly ideal molecular sieving support for the high-pressure gel filtration chromatography of proteins and was superior in performance to many currently available supports.

Factors affecting the relationship between the plate height and the linear mobile phase velocity in gel filtration chromatography of proteins

Factors affecting the relationship between the plate height and the linear mobile phase velocity in gel filtration chromatography of proteins

Scaling up of medium-performance gel filtration chromatography of proteins.

Since gel filtration chromatography with particles having a relatively small diameter and high mechanical stability (medium-performance gel filtration chromatography, MPGFC) is useful not only in scaling up the separation of proteins buj also in reducing the separation time, HETP of proteins and pressure drop were measured for MPGFC (particle diameter, 44μm) columns of various column dimensions. The smallest column was 1.0cm in diameter and 30cm in length (24cm3), while the largest was 9 × 90cm (5726cm3). The dependences of HETP and of pressure drop per unit length on column dimensions were found to be negligible for these columns. The sample volume dependence of HETP was predicted well on the basis of the HETP at small sample volumes, when the protein concentration is low. However, when the protein concentration was increased to above 3%, HETP increased sharply. This increase of HETP was considered to be due to the relative difference between the viscosity of the sample and that of the elution buffer. From the above results, we expect that the scaling up of MPGFC of proteins can be easily carried out on the basis of the data obtained with a small column.

Physical properties of collagen--sodium dodecyl sulfate complexes.

Sodium dodecyl sulfate (NaDodSO4)--polyacrylamide gel electrophoresis and gel filtration chromatography of protein--NaDodSO4 complexes are frequently used to characterize collagen-like polypeptide components in mixtures obtained from extracts of basement membranes. However, electrophoresis yields anomalously high apparent molecular weights for collagenous polypeptides when typical globular proteins are used as molecular weight standards, and the use of gel filtration chromatography for this purpose was suspect because Nozaki et al. [Nozaki, Y., Schechter, N. M., Reynolds, J. A., & Tanford, C. (1976) Biochemistry 15, 3884--3890] found that asymmetric particles, including NaDodSO4--protein complexes, coeluted with native globular proteins of lower Stokes radius, when Sepharose 4B was used. To understand these effects and to improve the characterization of collagenous polypeptides, we investigated the secondary structure of NaDodSO4--collagen complexes with the use of circular dichroism, measured the NaDodSO4 content, studied the dependence of electrophoretic mobility on gel concentration, and extended work on gel filtration by use of a more porous gel, Sepharose CL-4B. We found that the anomalous behavior of collagen chains on NaDodSO4--polyacrylamide gel electrophoresis is due in large part to treatment of data and that the method can be used to determine rather accurate values for the number of residues per polypeptide chain. Our gel filtration results indicated that reliable molecular weights can be obtained when Sepharose CL-4B is used. These methods can be applied equally well to collagenous and noncollagenous polypeptides.