Protein chromatography molecular weight determination

Fractionation of proteins according to size utilizing cross-linked dextran or polyacrylamide gel columns was first demonstrated by Porath and Flodin 63 in 1959. This technique has become the most widely accepted method for separation and molecular weight determination of hydrophilic and some hydrophobic macromolecules using aqueous buffers with or without organic modifier. While this technique might not be unique in its ability to resolve and separate proteins, it is one additional simple and effective tool in the chemist s armamentarium. The theories behind size-exclusion HPLC and size-exclusion chromatography at low pressure are identical and are described in several publications. 31 34 36 39 44 64 65 ... 

Molecular Weight Determination. The molecular weight of a protein is an important criterion. It provides the idea about the relative size of the protein molecules. The molecular weight is traditionally determined by ultracentifugation or by chromatography through a matrix... 

The spectrum in Fig. 4.9 demonstrates that MALDI-TOF is a powerful method for accurate molecular weight determination of peptides and proteins. As there is almost no fragmentation, mixtures of peptides and proteins can be analysed without having to separate the compounds prior to analysis. In this respect, MALDI-TOF has to be regarded as a very fast separation method and is in many ways more powerful than chromatography or electrophoresis. In Fig. 4.10, a spectrum of low fat bovine milk is shown. The milk sample was added to the matrix without any pre-treatment and the different components present in the sample are resolved in the obtained mass spectrum. 

Effective hydrodynamic radius, R, dictates solute partitioning in a gel. This can be related to the number of units in a flexible polymer. It is then a corollary that partitioning of a linear polymer is a function of Its molecular weight. This has been proved experimentally leading to popularization of gel chromatography in 6M guanidinium chloride as a useful method of protein molecular weight determination. 

A new protein of unknown structure has been purified. Gel filtration chromatography reveals that the native protein has a molecular weight of 240,000. Chromatography in the presence of 6 M guanidine hydrochloride yields only a peak for a protein of M, 60,000. Chromatography in the presence of 6 M guanidine hydrochloride and 10 mM /3-mercaptoethanol yields peaks for proteins of M, 34,000 and 26,000. Explain what can be determined about the structure of this protein from these data. 

The cell-bound amylopullulanase was solubilized with detergent and lipase. It was then purified to homogeneity by treatment with streptomycin sulfate and ammonium sulfate, and by DEAE-Sephacel, octyl-Sepharose and puUulan-Sepharose column chromatography (12). The final enzyme solution was purified 3511-fold over the crude enzyme extract with an overall recovery of 42% and had a specific activity of 481 units/mg protein. The average molecular weight of the enzyme was 136,500 determined by gel filtration on Sephacryl S-200 and SDS-PAGE, and it had an isoelectric point at pH 5.9. It was rich in acidic and hydrophobic amino acids. The purified enzyme was quite thermostable in the absence of substrate even up to 90°C with essentially no loss of activity in 30 min. However, the enzyme lost about 40% of its original activity at 95 C tested for 30 min. The optimum tenq)erature for the action of the purified enzyme on pullulan was 90°C. However, the enzyme activity rapidly decreased on incubation at 95°C to only 38% of the maximal 30 min. The enzyme was stable at pH 3.0-5.0 and was optimally active at pH 5.5. It produced only maltotriose and no panose or isopanose from pullulan. 

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