Disk electrophoresis

Some properties of Penicillium fellutanum pectinesterase were studied. The optimum of pectinesterase action was detected at pH 5 and 45 °C. The enzyme was stable at pH 4 — 5 and 40 °C (pH 5) for 240 min. and was specific towards lemon pectin. An enzyme preparation composed mainly of pectinesterase was partially purified by gel filtration. Pectinesterase activity was accumulated in one of the obtained fractions. Molecular weights of fraction determined were found to be 46,000 and 1,200. Disk electrophoresis in polyacrilamide gel of the purified preparation revealed two protein bonds with one active component. The partially purified enzyme had the kinetic characteristics =... 

Miller and Macmillan [4] carried out purification of pectinesterase from Fusarium oxysporum f. sp. vasinfectum culture fluid (fivefold degree of purification). According to the obtained data the purified enzyme possessed very low polygalacturonatlyase one. Disk electrophoresis at pH 4.3 revealed two protein components. The authors did not study distribution of pectinesterase activity in these components. Molecular weight of fungal pectinesterase determined using gel — filtration on Sefadex G — 75 was found to be 35,000. 

Molecular weight of the components of the enzymatic complex was determined using a Sephadex G —75 column after its calibration by dextrans with molecular weight equal to 10,000, 40,000 and 70,000 and rafinose with molecular weight of 504. Fractions were also analyzed by the disk —electrophoresis method in PAAG (7) using 7.5% polyacrilamide gel (pH 4.3). Activity of pectinesterase was determined by titrometric method [8]. The enzymatically released methanol analyzed by gas—liquid chromatography [9]. 

Disk electrophoresis of pectinesterase enzyme preparation in polyacrilamide gel revealed 4 protein bands in toe fraction 1 after its gel — filtration through Toyopearl HW—55. One of the two obtained bands (a wider one) possessed pectinesterase activity, while toe second one did not reveal it. 

Adsorption of the eel enzyme to the affinity column was quantitative and approx. 80% of the activity was recovered after elution with the inhibitor tensilon. Specific activity of the enzyme which migrated on disk electrophoresis as a single band was 16 000 units/mg. Results with the erythrocyte enzyme were equally good, with a 2500-fold purification achieved. 

Figure 2 Set-up for disk electrophoresis. (A) Tree-layer polyacrylamide gel ready for the run (B) isotachophoretic sample component stacking during the first few minutes of run (the process starts in the sample gel and has to be completed when leaving the spacer gel) (C) progress of separation in the running gel (Reproduced from Ornstein L (1964) Annals New York Academy of Sciences 121 321-349).

Kasparov, S. Y., F. A. Tikhomirov, and A. D. Fless. 1981. Use of disk electrophoresis to fractionate humic acids. Soviet Soil Science 36 21. 

Disk electrophoresis on polyacrylamide gels of the denatured enzyme-[ S]MMPR-OP complex is carried out to determine the subunit bound by the affinity label. ... 

FIGURE 13.15 Disk electrophoresis apparatus for gel isoelectric focusing. Q> sample. 

Marx P, Kovach J (1966) In Current topics in developmental biology. Academic Press, London New York, pp 213-252 Marzluf G (1965) Genetics 52 503-512 Massaro E, Markert C (1968) J Exp Zool 168 223-238 Masters C, Hinks M (1966) Biochim Biophys Acta 113 611-613 Masters C, Holmes R (1972) Biol Rev 47 309-361 Masui I (1972) Ontogenesis (Russ) 3 574-587 Masui I, Markert C (1971) J Exp Zool 177 129-146 Matsuzawa T, Hamilton J (1973) Proc Soc Exp Biol Med 142 232-236 Matveeva NM, Korochkin LI (1974) Ontogenesis (Russ) 5 92-95 Maurer G (1971) Disk-electrophoresis. Mir, Moscow Maximovsky LF (1970) Cytologia (Russ) 12 887-893... 

Slab gel, column and disk electrophoresis (Topic D8) are the principal formats. They allow multiple samples and standards to be run simultaneously for comparison purposes, which is comparable to separations by TLC (Topic D3). Samples are placed in wells near the centre of horizontal slabs when both cationic and anionic species are to be separated, or at one end if all solutes are expected to carry the same charge. With vertical slabs, samples are placed in wells at the top of the slabs so that only downwards migration is possible. 

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