By Ronald L. Nagel
A set of comfortably reproducible experimental and scientific ideas for learning the molecular foundation of hemoglobin issues. those step by step tools make the most of a large choice of the newest analytical recommendations, together with x-ray crystallography, excessive functionality liquid chromatography, electrophoresis, and nuclear magnetic resonance. extra equipment are provided for prenatal diagnostic research, the DNA analysis of hemoglobin mutations, hemoglobin fluorescence, and the semisynthesis of hemoglobin.
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Extra info for Hemoglobin Disorders: Molecular Methods and Protocols (Methods in Molecular Medicine)
Other columns or techniques may nevertheless be more appropriate for some specific separations. When chromatographic methods are used for globin chain quantification, it is important to consider the absorption coefficient of the various chains at the wavelength of detection. In some cases, it may be identical, such as when comparing the various γ-chains. In other cases, the absorption may differ considerably; for example, at 280 nm, γ-chains, because of their 3 Trp residues, have a higher ε coefficient than β-chains (2Trp) and α-chains (1 Trp).
1990) Structure and function of hemoglobin variants at an internal hydrophobic site: consequences of mutation at the beta 27 (B9) position. Biochemistry 29, 7020–7023. 34. Moo-Penn, W. , Jue, D. , Johnson, M. , Olsen, K. , Jones, R. , Lux, S. , and Arnone, A. (1988) Hemoglobin Brockton [β138(H16) Ala → Pro]: an unstable variant near the C-terminus of the b-subunits with normal oxygen-binding properties. Biochemistry 27, 7614–7619. 35. , and Bonaventura, C. (1980) Structural and functional studies of hemoglobin Suresnes (Arg 141α2 → His α2): consequences of disrupting an oxygen-linked anion-binding site.
28 Wajcman 3. In my laboratory, for routine determination of the γ-chain composition, we replaced this procedure with an RP perfusion chromatography using a Poros R1® column (Applied Biosystems) (14). 4. To obtain good reproducibility, we recommend using the same glassware for preparing the solvents. Solvents may be kept refrigerated at 4°C for a few days. Accurate balance of the TFA between both solvents is important to avoid baseline drift. Acetonitrile must be of HPLC grade with low UV absorbancy in the 210-nm region.