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Effect of urea on pI's of ampholytes and casein in isoelectric focusing☆

The isoelectric point (pI) of human red cell glucose transporter (Glut 1) was determined. Inconsistent values of 6.0, 6.4–6.5 and 8 have been reported earlier. Integral membrane proteins from human red cells were analyzed by two-dimensional electrophoresis with isoelectric focusing and sodium dodecyl sulfate gel electrophoresis (2D-PAGE). A zone of monomeric Glut 1 was found at pH 8.7, but most of the Glut 1 focused at pH 6–7 together with the anion transporter and other components. Purified Glut 1 focused only at pH 8.5 ± 0.2 (S.D., n = 12) and deglycesylated purified Glut 1 only at pH 8.4 ± 0.1 (n = 5), as shown by 2D-PAGE. The absence of Glut 1 below pH 8 in the latter cases was confirmed by immunoblotting with a monoclonal antibody. Furthermore, Glut 1 was photoaffinity-labelled with [³H]cytochalasin B and subjected to isoelectric focusing in one dimension. The pI of the labelled Glut 1 was 8.6 ± 0.3 (n = 11). A pI of 9.1 was calculated for the Glut 1 polypeptide on the basis of amino acid composition and pK_a values for amino acid side groups. The sialic acid content of the glycosylated transporter from fresh red cells was determined at approximately 2.1 sialic acid residues per transporter, which corresponds to a calculated pI of 8.8. The pI values of other human glucose transporter polypeptides of the facilitative diffusion type (Glut 2, 3, 4 and 5) were calculated at 8.4, 7.4, 7.1 and 6.2, respectively.

It is possible to measure pH values in immobilized pH gradients (IPG) when the polyacrylamide matrix is made to contain an additional, carrier ampholyte-generated pH gradient. After an IPG run, 5 mm gel segments, along the separation axis, are cut and eluted in 300 μl of 10 mM KCl and the pH read with a standard pH meter. When using ultranarrow pH gradients, larger gel segments (ca. 265 μl) are eluted in 900 μl of 100 mM KCl and the pH assessed with a differential pHmeter. In the latter case, either internal or external standards are used as a reference, or starting point, to convert Δ pH values into an actual pH curve. The reproducibility of the system is better than ±0.05 pH units, with a ca. 15% error over a 0.3 pH unit span. In ultranarrow pH gradients, it is imperative to use mixtures of all commercially available carrier ampholytes, so as to smoothen conductivity and buffering capacity gaps. By the present method, it is also possible to convert a wide (2–3 pH unit) carrier ampholyte interval into a narrow (0.2–0.3 pH unit) one.

It is impossible to measure pH values in immobilized pH gradients either with a surface electrode or by cutting gel slices and eluting in 10 mM potassium chloride solution. The use of reversible gels, cross-linked with bisacrylylcistamine, improves the measurements in the acidic region, but gives false values in the alkaline region, owing to the buffering power of added 2-mercaptoethanol or dithiothreitol. By using mixed-type gels, containing Immobilines and 1% carrier ampholytes, accurate and reliable pH measurements can be obtained. The discrepancy between the theoretical slope of the immobilized pH gradient and the actual pH values obtained by reading the pH of eluted Ampholine focusing in the same gel fragment is less than 0.1 pH unit over a 1 pH unit span. The effects of temperature and of carbon dioxide absorptionbnd on pH readings are demonstrated and evaluated.

Citraconic anhydride reacts readily with α-crystallin's lysine residues at pH 7.4. Upon addition of 2 equivalents of citraconic anhydride per equivalent lysine, 24% of the lysine residues were modified without disrupting the native quaternary structure. Further citraconylation led to dissociation into 10 S aggregates. Complete dissociation into subunits (1.4 S) occurred after adding 100 equivalents of citraconic anhydride, resulting in 98% modification. Decitraconylation did not lead to reaggregates identical with the native ones. The unmodified and the once and twice citraconylated α-crystallin subunits were discerned by isoelectric focusing according to their theoretical isoelectric points. In the native α-crystallin aggregates, nearly all B chains and approx. 60% of the A chains were found to possess at least one surface-exposed lysine residue. No differences between the susceptibilities to citraconylation of the in vivo deamidated (A₁ and B₁) and the de novo synthesized (A₂ and B₂) subunits were found. These results support the three-layer spherical assembly model for the α-crystallin quaternary structure.

The four subunits of α-crystallin, αA₂, αA₁, αB₂, and αB₁, have been separated on a preparative scale by application of the novel method of chromatofocusing in 6 m urea. Analytical polyacrylamide gel electrophoresis revealed the purity of the isolated subunits. The recovery was almost 75%.

Immunoglobulin IgM₂₂, secreted by mouse plasmacytoma ABPC-22, was characterized on the basis of ligand binding and physicochemical properties. Upon purification, only two polypeptides (μ aqnd κ chains) were detected by 6 M urea isoelectric focusing in polyacrylamide and SDS-polyacrylamide electrophoresis (SDS-PAGE). The μ and κ chains possessed pI values of 6.27 and 6.13 respectively, in 6 M urea. The molecular masses of μ and κ chains were established by SDS-PAGE and molecular sieve chromatography to be 70 and 30 kd, respectively. Thus, a molecular weight of 1 × 10⁶ was estimated for non-reduced IgM₂₂, and was confirmed by molecular sieve chromatography and sedimentation equilibrium ultracentrifugation. J chain was detected in freshly purified samples. As shown by competitive hapten inhibition (CHI) of the precipitation assay, using ¹²⁵I-rhodamine B₅BSA as the reference ligand, IgM₂₂ bound a wide range of structurally related and unrelated probes. Competing for the same site were polyaromatic anions (such as pyrene-1-sulfonate, rhodamine B and fluorescein) and weakly inhibitory organic acids (such as acetate and propionate). In addition. Fab fragments inhibited precipitation of ¹²⁵I-rhodamine B₅BSA by IgM₂₂. Equilibrium dialysis studies revealed a K_af 1.5 × 10³M⁻¹ for the interaction of ¹⁴C-fluorescein with IgM₂₂. A K_a of 3.2 × M⁻¹ for rhodamine B was determined using a molecular sieve column equilibrated in varying concentrations of rhodamine B. Based on relative K_a values, CHI results and fluorescence binding data with anilinonaphthalene sulfonate (ANS), the nature of the antibody active site was described in detail.