Electrophoresis starch

It has been shown (37) that the minimal molar ratios per 100 moles of aspartic acid found in the 16 proteins prepared by starch electrophoresis of the sera of dog, rat, and man were glu 90, ala 70, thr 50, ser 40, and gly 30. This represented the hypothetical A peptide. When these ratios were subtracted from the values of the molar ratio of each amino acid from each protein, the values given in Table m were found. Peptide B is represented by the excess number of moles of ala plus glu, which equals n in Equation 1 peptide C is represented by the values from excess gly plus ser. The sum of these two values for each protein is equal to K, and... 

G26. Grasbeck, R., Fractionation of human gastric juice and saliva employing starch electrophoresis. Gastroenterologia 84, 99-102 (1955). 

Normal individuals excrete 31 mg of albumin in their urine daily. The studies of the physical and chemical properties of the urinary albumin have established that the urinary protein is identical to the blood protein. Both serum and urinary albumin have the same mobilities on free-starch electrophoresis and im-muno-electrophoresis. The sedimentation constants and the diffusion rates of the two proteins are similar, and their molecular weight must be of the order of 73,000. The two proteins react identically antigeni-cally. 

SwAHN (1953) and Carlson and Olhagen (1954) found that lipemic plasma subjected to free and starch electrophoresis respectively, contained two turbidity peaks. Increases in lipids migrating as jS-lipoproteins on starch have been reported by Dietrich (1955) and by Schettler et al. (1958), who also observed a significant decrease of this fraction after preliminary ultracentrifugation and separation of the chylomicron fraction. Paronetto et al. (1957) described increases in ol -lipoproteins on starch electrophoresis. Schettler et al. (1958) described disappearance of chylomicrons in this area on low fat diets. 

Paronetto, F., C. Wang, and D. Adlersberg Lipoprotein patterns by starch electrophoresis in idiopathic hyperlipemia and hypercholesteremia. Circulat. Res. 5, 288 (1957). 

Cathepsin D (from bovine spleen) [9025-26-7] Mr 56,000, [EC 3.4.23.5]. Purified on a CM column after ammonium sulfate fractionation and dialysis, then starch-gel electrophoresis and by ullracentrifugal analysis. Finally chromatographed on a DEAE column [Press et al. Biochem J 74 501 I960],... 

Ferguson, KA, Starch-Gel Electrophoresis— Application to the Classification of Pituitary Proteins and Polypeptides, Metabohsm 13, 985, 1964. 

Fig ure 3. Starch gel electrophoresis of hemoglobins. Tris-EDTA-boric acid buffer, pH 9.0. O-Dianisidine stain. 

Att eZcven y-cJuiin voA nts, discovered thus far, exhibit a change In electrophoretic mobility, and starch gel electrophoresis Is the recommended method for their detection. Quantitation of the variant can best be done by chromatography on columns of either DEAE-Sephadex or CM-Cellulose. The quantities of some variants In heterozygotes differ greatly. For Instance, the relative amount (expressed In %F /Fxotal) varies from 20-25% (F-Malta-I) to 10-15% (most Y C >aln variants) to 5-6%... 

The, chain voAiantS are characterized by the presence of two abnormal components, an abnormal Hb-F (02 /2) and an abnormal Hb-A (tt2 32) Of these two, the 02 2 component dominates and the 02 32 component Is often difficult to detect. The methods of choice are starch gel electrophoresis and anion-exchange chromatography using DEAE-Sephadex or DE-52 Cellulose. Chain analyses of these Isolated hemoglobin components will lead to a definitive Identification. 

The defnon6ttLOtion 0 -chain vaAijant6 In heterozygotes Is complicated by the presence of the large amount of Hb-F. Another obstacle Is the nearly Identical electrophoretic mobilities of Hb-A and the minor Hb-Fi component. Despite these difficulties, abnormalities such as AS, SS, AC, CC, SC, and others can readily be detected using cellulose acetate electrophoresis, starch gel electrophoresis, acid agar electrophoresis, and by CM-Cellulose microchromatography to be described In a separate section. 

Hb-B0Jut 6 OK yif can best be demonstrated by either cellulose acetate or starch gel electrophoresis. The amount of Hb-Bart s can differ from 1% to over 80% dependent on the abnormality Involved. Quantitative determination Is most accurately made by CM-Cellulose or CM-Sephadex chromatography. 

Hb-A2 can also be quantitated by electrophoresis. The most accurate procedures are starch block electrophoresis, cellulose acetate electrophoresis, and isoelectric focusing. 

Mlcrochromatographlc analyses were made In Ghana, and starch gel electrophoresis In the USA. 112 samples lost In transport 145 samples not mailed 88 samples not Identifiable. 

The validity of diagnosis by this technique has been examined by comparing more than 2,000 samples by the CM-Cellulose procedure, the original CM-Sephadex procedure, and by starch gel electrophoresis. It Is Interesting that occasionally the AS condition at birth Is not diagnosed by the electrophoretic technique. 

Free a-and 3-chains can be demonstrated In a freshly prepared hemolysate using starch gel electrophoresis at alkaline pH. 

Detection of Met(Ferrl-)Hemoglobins (Hb-M) Detection of these variants can be made by starch gel electrophoresis of the ferrl-derlvatlves of hemoglobins In red cell hemolysate using a phosphate buffer, pH 7 0 (25) However, some methemoglobln variants can be separated from normal Hb-A at pH 9 0 (40) ... 

The presence of Individual chains In a hemoglobin variant can also be demonstrated by electrophoresis at alkaline pH after the protein has been dissociated Into Its subunits through exposure to 6 M urea In the presence of 3-mercaptoethanol. The buffer is either a barbital buffer or a tris-EDTA-boric acid buffer, pH 8.0 - 8.6, and contains 6 M urea and 3-niercapto-ethanol. Dissociation of the hemoglobin Into subunits Is best accomplished In a mixture of 1 ml 10 g% Hb (or whole hemolysate), 4 ml 6 M urea barbital or tris-EDTA-boric acid buffer, and 1 to 1.5 ml 3-mercaptoethanol. After 30 minutes to 1 hour the sample Is subjected to cellulose acetate or starch gel electrophoresis. Each chain has a specific mobility and an alteration In electrophoretic mobility easily Identifies the abnormal chain. 

By means of gel electrophoresis on cross-linked, hydrolyzed starch,99 with simultaneous checking for proteins, lipids, and pectinesterase activity, it was found, however, that the product isolated after the separation on CM-Sephadex C-50 constitutes but one of five multiple forms of tomato pectinesterase, and is the one present in preponderant proportion98 (see Fig. 4). The accompanying lipid and sugar components were separated from this pectinesterase form in the course of the purification procedure. After analysis of the hydro-lyzate of the final product for fatty acids, as well as for carbohydrate components, it was possible to exclude the possibility of a lipoprotein,30 as well as glycoprotein,100 character of this form of tomato pectinesterase. 

Fig. 4. — Monitoring of the Multiple Molecular Forms of Tomato Pectinesterase by Starch-gel Electrophoresis.98 [ENZ, detection of pectinesterase activity by paper print with pectin and Bromothymol Blue PROT, protein staining with nigrosin O, origin. Key A, 1 crude tomato extract after ammonium sulfate salting-out, and dialysis 2 pectinesterase fraction from column of DEAE-Sephadex A-50 3 and 4 pectinesterase fractions from column of Sephadex G-75. B, Two parts of the same gel after horizontal slicing 1, 500 fig of the isolated form of pectinesterase from a column of CM-Seph-adex C-50 with 175 mM phosphate-sodium chloride buffer 2, active fraction at 150 mM buffer 4 and 5, 250 fig and 1 mg of the isolated form of pectinesterase, respectively.]...

By use of starch-gel electrophoresis, the total extract of bananas, and the fractions obtained after separation on DEAE-Sephadex A-50, were found to contain six multiple forms of pectinesterase having electrophoretic patterns different from those of tomato pectinesterase.103... 

The pectinesterase produced by Sclerotinia libertiana78 was purified on columns of Duolite A-2, Amberlite CG-50, and CM-cellulose. The final product was purified 266-fold, its sedimentation coefficient was calculated to be 4.41 S, and zone electrophoresis in starch gel showed a slight contamination of this product. 

The only pectic enzyme thus far obtained in crystalline form is the endo-D-galacturonanase from Acrocylindrium.209 Crystallization of the enzyme from a solution of ammonium sulfate was preceded by chromatography on calcium phosphate, Duolite CS 101, and DEAE-cellulose, and by starch-gel electrophoresis. 

Fig. 1. Schematic presentation of the protein pattern of the common Hp types in pure form after starch-gel electrophoresis. The protein pattern of a normal serum belonging to Hp type 1-1 is given at the bottom. The cathodic part is excluded.

It was soon realized (J10, R5) that Hp did not consist of a single protein, but of a group of proteins with very similar properties. Conclusive evidence of the molecular heterogeneity of Hp was produced by Smithies (S5), who used electrophoresis with a defined starch gel in which the mobility of protein molecules varies with their charge and size (Fig. 1). Smithies and Walkers discovery (S9) of different types of... 

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