Starches starch gels

Electrophoretic methods using supporting media, such as starch, starch-gel, cellulose acetate, agar gel, continue to be of importance in detecting abnormal hemoglobin variants. The techniques are less suit-... 

Starch Gels The first gel media to receive attention was starch. Starch gels are prepared by heating and cooling a mixture of partially hydrolyzed starch in an appropriate buffer. This causes the branched chains of the amylopectin component of the starch to intertwine and form a semi-solid gel. 

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. 

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. 

Zhang, B.J., Davis, S.A. and Mann, S. (2002) Starch gel templating of spongelike macroporous silicalite monoliths and mesoporous films. Chemistry of Materials, 14, 1369-1375. 

Pure starch is not much used in bakery products. Some maize starch goes into a few types of biscuits or cakes under the heading of corn flour . A few starch gels are applied on bakery flans. 

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. 

By use of starch-gel electrophoresis175,195 and disc electrophoresis196 and various separation methods, D-galacturonanases were found to occur as multiple molecular forms.167,173-175,184,197... 

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... 

The saturated HpHb complex of type 1-1 was formed in solution and mixed with a pure solution of globin. The proteins were then separated by starch gel electrophoresis with a neutral phosphate buffer. The procedure was afterward repeated, but with a solution of Hp-globin complex mixed with an Hb solution. 

Several modifications of Smithies original Hp-typing technique (S5) have been proposed. It has generally been accepted as simplest and safest to identify the Hp by its saturated Hb complexes (HpHb), since these are more easily recognized and developed than Hp itself in starch gel and in immunoelectrophoresis. To convert all Hp to HpHb before the analysis, more Hb is added to serum than can be bound by the Hp. 

Smithies vertical starch gel electrophoresis (S7) separates the plasma proteins more distinctly than any other method. If the Hp concentration is normal, the Hp type can generally be recognized directly after the staining for proteins, but sensitive and more specific staining for heme groups, e.g., benzidine, o-dianisidine (04), and malachite green (N5) are preferable. This technique consumes more hydrolyzed starch than the simpler original horizontal electrophoresis technique (S5). 

The Hb solutions generally used are obtained by simple osmotic hemolysis of normal red cells followed by elimination of the ghosts. The molecular heterogeneity of such solutions of adult Hb is revealed by the starch-gel electrophoresis. The Hb line is therefore not quite distinct, which is a minor drawback when the solutions are used for Hp typing. To stabilize the Hb solutions, it is advisable to bubble CO through them before they are ampouled and stored in the frozen state. 

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