Tiselius apparatus

The soluble protein showed a single boundary peak in the Tiselius apparatus in buffers of ionic strength 0.02 at all pH values in the range pH 2-9, but its isoelectric point was markedly dependent upon the salt concentration. At ionic strength 0.2, in the presence of sodium chloride, the isoelectric point both from electrophoretic mobility measurements and membrane potential determinations was pH 3.9-4,0. At lower ionic strength (0.02) the protein was isoelectric at pH 4.8 in the electrophoresis experiments and pH 4.7 in membrane potential determinations. 

The Tiselius Apparatus.—The U-tube portion of the apparatus, shown in section (I) and in plan (II) in Fig. 130, is in several parts which can be slid horizontally across one another along the planes oa, 66 and cc the surfaces between the sections are suitably lubricated in order to facilitate the sliding and to prevent leakage of liquid. The channel running through the center of the apparatus, shown by the thicker lines,... 

Not only can the Tiselius apparatus be used to indicate the presence of several constituents in a system, but an actual separation of different fractions is sometimes possible. If sufficient time is allowed for the leading boundary to get some distance ahead of the next, the solution between it and the following boundary will consist almost entirely of the faster moving constituent. Since electrophoretic mobilities are, in general, not very different, the boundaries will have moved above the section C or below B in Fig. 131 before there is any appreciable distance between them. If the solution as a whole is given a velocity equal to that of the slower moving boundary but opposite in direction, this boundary will remain stationary at bb in Fig. 131 while the faster one moves steadily ahead, e.g., from 6 into section C. After some time the section C can be... 

Fig. 2. pH Activity curves of two anodal-moving components separated from concentrated human gastric juice in the Tiselius apparatus. , Major, faster component, mobility 2.99 X 10— cm /volt/sec o, minor, slower component. Electrophoresis was carried out in 33 mM acetate buffer, pH 2.5. Protein concentration, 0.51 g/100 ml. Human serum albumin as substrate temp. 37° time 3 hr. From Taylor (T15). 

Merten et al. (M4) precipitated HCl extract of hog gastric mucosa by 60% saturation with ammonium sulfate below pH 6.0 and, after dissolving, submitted it to paper electrophoresis at pH 4.6. The most anodic protein fractions contained proteases with a pepsin to cathepsin ratio of 10.9 1, while the second peak contained both these enzymes at a ratio of 1.6 1. After re-electrophoresis, the first peak demostrated peptic activity only at pH 1.4-2.4, with no activity at pH 3.5. Prolonged electrophoresis in the Tiselius apparatus of crystalline hog pepsin showed heterogeneity of this relatively pure material in acetate and phosphate buffers of pH 3.9, 5.9, and 8.0 (HIO). 

A more complete diagram of Tiselius apparatus (modified in details by Longsworth) is shown in Fig. 5. The vessel A-A is attached, as... 

The utility of the Tiselius apparatus in revealing the presence of closely related substances is indicated in Fig. 7, which is from a series of photographs of boundaries in the channel a-a of Fig. 4 made visible... 

Another purified enzyme preparation which produces laminaripentaose from insoluble laminarin and from heat-treated pachyman is produced by a strain of Arthrobacter luteus (100,101,102) when grown on yeast cells or / -(1 —>3)-glucan. The enzyme, which was named Zymolase (also referred to as Zymolyase) appeared to be homogeneous by electrophoresis in a Tiselius apparatus and by ultracentrifugation. The molecular weight of the enzyme was estimated from ultracentrifugation to be ca. 20,500. The optimum pH for lysis of viable yeast cells was 7.5. The optimum temperature was 35°C. The optimum pH for heat-treated pachyman hydrolysis was 6.5, and the optimum temperature was 45°C. A Lineweaver-Burk plot with heat-treated pachyman yielded a Km value of 0.04% when the solubilized carbohydrate was assayed by the phenol-sulfuric acid method. Zymolase lost all its activity after incubation at 60°C for 5 min. 

Moving Boundary Electrophoresis An indirect electrophoresis technique for particles too small to be viewed. This principle is used in the Tiselius apparatus. Here a colloidal dispersion is placed in the bottom of a U-tube, the upper arms of which are filled with a less dense liquid that both provides the boundaries and makes the connections to the electrodes. Under an applied electric field the motions of the ascending and descending boundaries are measured. 

Tiselius Apparatus An apparatus for the determination of electrophoretic mobilities. See Moving Boundary Electrophoresis. 

From 1945 on, Dubuisson has examined electrophoretically in the Tiselius apparatus extracts of muscle prepared with extracting media of differing salt content. By this means, he found three myosin components, a, /3 and y. It is almost certain that /3-myosin is identical with L-myosin and a-myosin with actomyosin (Table V). 7-Myosin seems to be identical with contractin (cf. Section IV, 3). The Y-protein may... 

The ratio of L-myosin to F-actin can be estimated quantitatively only from the concentration gradient curves in the ultracentrifuge or Tiselius apparatus (Dubuisson, 1946b). 

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