Hemoglobin Electrophoretic mobility

The techniques mentioned here provide a basic approach to the study of hemoglobinopathies since the Identification of many variants Is dependent upon their net charge. However, certain changes In the tertiary structure of a hemoglobin variant may also alter Its electrophoretic behavior. This Is well documented for many unstable variants such as for Hb-K51n which migrates more slowly than Hb-A but In which the neutral valyl residue In position 398 Is replaced by the neutral methlonyl residue. It should also be mentioned that differences In the level of heme oxidation will cause differences In the electrophoretic mobility. 

St/iuctu/uit >cn o (vUon gcUmd eZactAopkoA,etic pAoca-duAe will aid In detecting an a chaln abnormality because such a variant will be present as two abnormal bands, one major abnormality (02 32) and one minor abnormality (02 62). The absence of the minor abnormality does not exclude an a-chaln abnormality since Its electrophoretic mobility may be similar to that of a known normal hemoglobin type. The electrophoretic pattern in a subject heterozygous for the 3 chaln abnormality Hb-S or Hb-D and for the 6-chain variant Hb-A2 resembles closely that of a person who Is heterozygous for a Hb-D that Is abnormal In the a-chaln. 

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. 

The second abnormality, Hb-Tak, is a chain variant with an electrophoretic mobility slightly faster than that of Hb-S. Hb-Tak amounts to about 40% of the total hemoglobin and has been found in two individuals from Northwestern Thailand. The chain is extended at the carboxy-terminal end by ten amino acid residues, which are in the following tentative sequence (FlO) ... 

The hemoglobin consists mainly of Hb-Bart s (74) and some Hb-H ijSi). Some papers (L18, L22) report the presence of a small amount of normal Hb-A, but this conclusion is based on electrophoretic mobilities only. More recent studies (T8) have offered evidence for the pres-... 

Rigas, D. A., Koler, R. D., and Osgood, E. E., A new hemoglobin possessing a higher electrophoretic mobility than normal adult hemoglobin. Science 121, 372-373 (1955). 

Table 20-3 lists the principal plasma proteins and their half-lives, pi, molecular weights, and preferred method of analysis the individual proteins are listed in the order of their electrophoretic mobilities in agarose gels at pH 8.6. These proteins are described later in this chapter other chapters in this book describe many more proteins enzymes (see Chapter 21) lipoproteins (see Chapter 26) hormones (see Chapter 28) and hemoglobin, fibrinogen, and other coagulation proteins (see Chapter 31). The interim consensus reference intervals for 14 plasma proteins are listed in Table 20-4, pending the publication of more definitive intervals. . 

It is apparent from the above discussion that many hemoglobins with different amino acid substitutions demonstrate identical electrophoretic mobilities. Methods that rely on differences other than net charge are needed to establish the identity of these hemoglobins. Definitive... 

As compared to HbA. Hemoglobins A2, F, Bart s and H have multiple amino acid differences from HbA, and their electrophoretic mobilities depend on the net effect of all these differences on their net charge at pH 8.4-8.6. 

Table 1 lists the principal plasma proteins according to their electrophoretic mobilities in agarose with relative molecular mass, reference values, functions, and properties, and the causes of elevated and decreased levels. Other proteins are considered in detail in other articles in this encyclopedia (e.g., enzymes, lipoproteins, hemoglobin, and fibrinogen and other coagulation proteins). 

Fig. 3-3. Relative electrophoretic mobility of some hemoglobins (paper electrophoresis, pH 8.6 veronal buffer)...

Native electrophoresis of pepsin and hemoglobin on 10% polyacrylamide gel carried out at 48 °C during 90 min, according to the Laemmli procedure, at pH 8.3 (Laemly 1970). Water solutions of all samples of enzyme (pepsin dissolved in water to final concentration of 2 mg/mL) were titrated with HCl to pH 2 and incubated at 37 °C, with addition of different concentrations of Al + ion (1, 5 and 10 mM). The samples were diluted with sample buffer in ratio 1 1 (v/ v) and applied on gel in volume of 20 pL. Visualization was performed with Commassie Brilliant Blue G-250 dye. The gels scanned and processed using Corel Draw 11.0 software package. Quantification of electrophoretic mobility of the molecule is carried out via Rs value, where it is defined by ... 

Kaueman, S. L. Kuchumov, A. R. Kazakevich, M. Vinogradov, S. N. Analysis of a 3.6-MDa hexagonal bilayer hemoglobin ffom Lumbricus terrestris using a gas-phase electrophoretic mobility molecular analyzer. Anal. Biochem. 1998, 259, 195-202. 

In newborn infants only about 20% of the hemoglobin (Hb) is of the normal adult type, Hb A, and about 80% of the fetal type, Hb F (Haurowitz, 1924). The Hb F disappears from the blood during the first 7 months after birth. It has been known for almost a century that human fetal hemoglobin is much more resistant than adult hemoglobin to denaturation by sodium hydroxide. The difference in the rate of alkaline denaturation is used to identify Hb F and to estimate its proportion in a mixture of Hb A and Hb F. At pH >7.1, Hb A has a higher electrophoretic mobility than Hb F. 

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