304 BIOCHEMICAL GENETICS 



Amido black 10B, 360 or light green. 48 If desired, either the stained or the heat- 

 denatured, unstained paper strips can be placed in a recording device to determine the 

 mobility and relative amount of hemoglobin present. Several sources of error are 

 inherent in any type of device chosen; these factors have been reviewed elsewhere. 712 

 Hemoglobin solutions studied are not pure; for example, they generally contain car- 

 bonic anhydrase and methemoglobin reductase. Staining procedures are not specific 

 for hemoglobin but rather stain proteins. It is necessary to prove by other means that 

 a stained peak, especially a minor one, is hemoglobin. A few additional reports, not 

 mentioned above, concerning the use of paper electrophoresis for the investigation of 

 hemoglobin variants in many mammals, are easily available. 153, 331, 392, 443, 656, 1039, 

 i34o. 1384 Population studies are reported in these papers; the inheritance of some of 

 the variant forms has been established. 



Conventional paper-strip electrophoresis is not an efficient technique for obtaining 

 large quantities of a pure fraction of hemoglobin from blood that contains more than 

 one type of hemoglobin. Continuous flow, paper-curtain electrophoresis 111 has been 

 successfully used to separate serum fractions, but reports were not found of its use for 

 isolation of hemoglobins. In our laboratory we found that hemoglobins are tightly 

 adsorbed to the curtain and that good resolution of the fractions is difficult unless they 

 are electrophoretically quite different. 



2. Starch-block electrophoresis: Starch-block electrophoresis is often used for 

 preparative procedures when larger quantities of an electrophoretically pure fraction 

 are needed than can be obtained by paper-strip electrophoresis. The technique was 

 first described by Kunkel and Slater. 739 It is perhaps less suitable for routine analysis 

 than is paper electrophoresis, although comparable electrophoretic patterns are obtained 

 with either technique. It has the advantage that large quantities, up to 1,000 mg., of 

 hemoglobin can be applied; moreover, adsorption of proteins on starch is less pro- 

 nounced than on paper, which facilitates removal of individual components by elution. 

 It should be mentioned that electrophoresis on sponge rubber, from which materials 

 can easily be removed by squeezing, has also been investigated, 882 but its usefulness 

 for isolation of hemoglobins has not been reported. The procedures for starch-block 

 electrophoresis have been set forth previously. 737 The starch block is usually prepared 

 by pouring a barely liquid paste of washed potato starch and buffer, usually barbital 

 buffer, 0.05-0.10 ionic strength, pH 8.6, into a mold (38 x 10 x 1.5 cm.). Blotting 

 paper is placed at each end of the tray to remove excess liquid and is retained until 

 no more buffer remains on the surface of the starch. A slot is made in the starch block 

 and the sample, as either a solution or a starch paste, is added with a pipette. 



Minor components (A 2 and A 3 ) of normal human hemoglobin were first isolated 

 by use of starch-block electrophoresis. 736 ' 740, 856 The technique has been used clini- 

 cally in the diagnosis of the Thalassemia trait, an inherited condition in which the 

 quantity of A 2 hemoglobin is elevated from normal values of 1.2-3.5 per cent to ab- 

 normal values of 4.2-6.8 per cent of the total hemoglobin. 429, 738 An increase in the 

 amount of A 2 hemoglobin above 3.5 per cent is usually diagnostic for Thalassemia, 



