Enzymes paper electrophoresis

The purification of glutamine cyclotransferase from papaya latex has been carried out by Messer and Ottesen (116). A batch procedure was used for the removal of impurities by passage of a papaya latex extract through a thin layer of carboxymethyl-Sephadex the active protein was separated by selective elution. Additional purification was achieved by chromatography on a column of carboxymethyl-Sephadex and by gel filtration on Sephadex G-100. The purified enzyme was homogeneous by the criteria of paper electrophoresis, ultracentrifugation, and gel filtration on Sephadex G-100 columns and chromatography on carboxy-methyl- or DEAE-Sephadex. The electrophoretic behavior of the enzyme indicates that it is a basic protein with an isoelectric point near... 

Enzyme and substrate Cleavage shown by high voltage paper, electrophoresis [Ref. (3)] V max K (M)... 

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). 

Many derivatives of histidine are not stable to acid hydrolysis and are not discussed here (however, see 2.12.2 for those that occur naturally in proteins). Brief mention should be made of the iodination of histidyl residues by HOI ( 3.7.2). The mono- and diiodohistidines can be identified and distinguished from the iodinated tyrosines by high voltage paper electrophoresis in 1 M formic acid (Roholt and Pressman 1972) after complete enzymic hydrolysis of the protein or peptide ( 2.11 Roholt and Pressman 1972). Quantitation and identification are facilitated by the use of radiolabeled reagent. 

As an example of the use of paper electrophoresis for the fractionation of oligonucleotides consider the fractionation of some decanucleotides obtained from MS2 viral RNA by T1 ribonuclease digestion. The specificity of the enzyme ensured that each oligonucleotide contained only one Gp residue and the decanucleotides were isolated by column chromatography ( 5.2.1.2). The oligonucleotide mixture was electrophoresed for 17 hr at 6 volt/cm in 0.02 M ammonia formate pH 2.7. At this pH the nucleotides, Ap, Cp, Gp, Up have net charges, q, of —0.08, —0.02, —0.66, 0.98 respectively. Thus the mobility of these decanucleotides should depend mostly on the ratio of Ap+Cp to Up in their composition. Good separation in the predicted order has been obtained (Rushizky et al. 1965). 

The two-dimensional version of cross paper electrophoresis resembles two dimensional separations with a buffer change before the second run. This version is applicable when one or both reactants are not pure compounds. Crossed electrophoresis has found its wide applicability in immunochemical studies, in studies about enzyme-substrate interactions, and in interactions of proteins with a number of low molecular weight compounds. 

A stoichiometric amount (0.1 mM) each of enzyme-bound FAD and C-u-L-lysine (u = uniformly labelled) was incubated anaerobically until FAD was fully reduced. After deproteinization the reaction mixture was subjected to high voltage paper electrophoresis and paper chromatography. Most of the radioactivity appeared at the area corresponding to piperidine 2-carboxylic acid (a-keto-c-aminocaproic acid) (Figure 7), and a significant amount of carbon dioxide was not detected. When the reaction mixture was aerated to reoxidize FAD and then deproteinized, the radioactivity was also found at the position of piperidine 2-carboxylic acid. 

Figure 7. Formation of piperidine 2-carhoxylic acid. The enzyme-bound FAD (0.1 mM) and C-u-L.-lysine (0.1 mM, 1,000,000 c.p.m.) were incubated anaerobically for 7 hours. After deproteinization by adding perchloric acid, an aliquot of the reaction mixture was subjected to hi voltage paper electrophoresis (2000 volts) at pH 3.4 for 1 hour...

Alginate lyase from Turbo cornutus acted on sodium alginate and resulted in a rapid decrease in viscosity and a gradual increase in reducing power of the substrate solution. The result of gel filtration of the reaction products indicated that uronic acid oligosaccharides were liberated as the final product. The fact that the enzyme attacked preferentially the D-mannuronate-rich moieties of the alginate molecule was demonstrated by an analysis with paper electrophoresis of the reaction products after acid treatment, which separated the uronic acid oligosaccharides from the uronic acid polysaccharides. 

Extracts of acetone-dried kidney were purified about 1600 times by ammonium sulfate and acetone fractionation and by paper electrophoresis. The purest preparations reveal only single components in both electrophoresis and ultracentrifuge studies. The purified enzyme appears to contain only amino acids and has a molecular weight near 300,000. 

Compounds are isolated by preparative paper chromatography on Whatman No. 3 MM paper. Chromatograms are developed in freshly made n-butanol/water/glacial acetic acid (5 3 2) as solvent system for 2-3 days. Although this system is very slow, a more convenient system was not found because of the alkaline lability of the compounds. Purity of isolated products can be checked by paper chromatography in 1 M ammonium acetate (pH 7.4)/ethanol (1 1) and by paper electrophoresis in 0.05 M potassium phosphate at pH 7.0. Analysis of oligonucleotides is best carried out by enzymic degradation with snake venom phosphodiesterase or ribonuclease Tg. ... 

Fig. 9. The reaction catalyzed by the sialytransferases acyl is either acetyl or glycolyl. The assay method for these transferases usually involves incubation of acceptor, enzyme, and radioactive CMP-slallc acid followed by high voltage paper electrophoresis in 1% sodium tetraborate (Roseman et

In 1952 Russell Hilmoe and I became interested in ribonucleases and phosphodiesterases. It was clear that new methods would have to be devised in order to study the mechanism of action of such enzymes. At about this time Markham and Smith published their work on cyclic-terminal nucleotides and the separation of nucleotides by paper electrophoresis. I was excited by their work and arranged to spend the year 1953 in Roy Markham s laboratory in Cambridge, England. This turned out to be a very profitable and enjoyable year. I worked not only with Roy Markham and Paul Whitfeld, but also with Dan Brown, who was in Lord Todd s Chemical Laboratory. 

The aminoaoid sequence of oaerulein was deduced essentially by degradation with two enzymes, chymotrypsin and subtilisin. The fragments obtained were fractionated by preparative paper electrophoresis and examined by further degradation, chemical and enzymatic. 

To further study the mode of inhibition of pepsin and other acid proteases by pepstatin, we tried to produce various pepstatin fragments and measure their kinetics of inhibition. These pepstatin fragments are produced by three methods (a) complete acid hydrolysis yields free statine and other free amino acids (b) partial acid hydrolysis yields a mixture of peptides and (c) enzyme digestion with a-lytic protease yields the tetrapeptide, valyl-statyl-alanyl-statine. Purification by high-voltage paper electrophoresis from the above mixtures gives four statine-containing products in sufficient yield to allow further study statine, alanyl-statine, valyl-statine, and valyl-statyl-alanyl-statine. The isolated products are acetylated and quantified, and the completeness of their acetylation is determined prior to the initiation of inhibition studies (10). 

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