Hemoglobin labeling

Petcu, L. C. Turcu, G. Rosoiu, N. Comparative study of certain ferric and ferrous derivatives of hemoglobin labeled with HPT. Rom. J. Biophys. 2002,12, 53-58. 

The 5-iodoacetamido derivative of fluorescein (5-IAF) has been used to label numerous proteins and other biomolecules, including actin (Plank and Ware, 1987), myosin (Aguirre et al., 1986), troponin (Greene, 1986), hemoglobin (Hirsch et al., 1986), and sulfhydryl-containing proteins separated by SDS electrophoresis (Gorman, 1984). 

CL reaction can be catalyzed by enzymes other than HRP (e.g., microperoxidase and catalase) and by other substances [hemoglobin, cytochrome c, Fe(III), and other metal complexes]. The presence of suitable molecules such as phenols (p-iodophenol), naphthols (l-bromo-2-naphthol), or amines (p-anisidine) increases the light production deriving from the HRP-catalyzed oxidation of luminol and produces glow-type kinetics [6, 7], The use of other enzymes, such as glucose-6-phosphate dehydrogenase [38-41], P-galactosidase [42], and xanthine oxidase [43-46], as CL labels has been reported. 

In your notebook, make a drawing of the observed banding pattern, labeling both the hemoglobin and cytchrome C. Explain, in relevant detail, what you can determine about the isoelectric points of these proteins and what might happen in the gel if the buffer pH were changed from 7.9 to 3.9. 

Rudolph AS, Klipper RW, Goins B, et al. In vivo biodistribution of a radiolabeled blood substitute Tc-labeled liposome-encapsulated hemoglobin in an anesthetized rabbit. Proc Natl Acad Sci USA 1991 88 10976. 

Figure 1 shows the Raman spectrum of Hb obtained with 406.7-and 413.1-nm excitation and the spectrum of monomeric, four-coordinate Ni protoporphyrin in aqueous micellar solution (9). Excitation at 413.1 nm is at resonance with the red component of the split Soret band of Ni-reconstituted hemoglobin at 406.7 nm the blue component of the Soret band is selectively probed. Comparison of the spectra shows that two sets of marker line frequencies exist. One set (labeled 4 in Figure 1) is enhanced by resonance with the blue Soret component the other set (labeled 5) is enhanced by excitation of the red Soret component. Thus, the shifts in the core-size lines in going from set 4 - 5 are -39 cm (i/-q at 1657 cm ), -20 cm cm ), and -34 cm 1 19 cm ). 

Fig. 12. Assignments of the proton resonances of the C-2 protons of histidyl residues at p2 and pi46 of human normal adult hemoglobin 250-MHz aromatic proton resonances of deoxy-Hb A, deoxy-Hb Deer Lodge (p2His— Arg), and deoxy-des-His-(pi46-deleted)-Hb A in 0.1 M Bis—Tris in D20 at pH 6.3 and 27°C. The resonance lines are labeled according to the notation of Russu et al. (1982). [From Ho and Russu (1981)].

Sassaroli, M., Bucci, E., Liesegang, J., Fronticelli, C. and Steiner, R.F. (1984). Specialized functional domains in hemoglobin dimensions in solution of the apohemoglobin dimer labeled with fluorescein iodoacetamide. Biochemistry, 23,2487-2491. 

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