Sickle hemoglobin is more fusogenic than normal hemoglobin at physiological pH and ionic strength conditions.

We used electron microscopy, quasi-elastic light scattering and static light scattering to show that human hemoglobin (Hb) interacts with bovine brain phosphatidylserine lipid vesicles and promotes vesicle fusion in an isotonic buffer at pH 7.4. The fusogenic properties of Hb were observed in both small unilamellar vesicles (SUVs) and large unilamellar vesicles (LUVs). A simple turbidity measurement method was used to follow increases in vesicle size (scattering diameter) as a function of time. For the first 3 h, upon incubation with oxygenated Hb, the scattering diameters of vesicles increased at a rate of 7.8 nm/h for LUVs. Continuous incubation with Hb led to complicated vesicle fusion, probably due to the oxidation products of Hb and lipid molecules. In the absence of both Hb and lipid oxidation, using Hb liganded with carbon monoxide, we obtained, for the entire 20 h incubation period, a fusion rate of 2.9 nm/h for LUVs. We also studied interactions between sickle Hb and vesicles under the same conditions and found that the vesicle fusion rates for sickle Hb were about 2 times faster than those for normal Hb. These results showed that sickle Hb exhibited more extensive interactions with lipid bilayer than normal Hb at physiological pH and ionic strength conditions, and provide insights toward understanding the molecular mechanisms in sickle cell abnormalities.

The first human alpha-spectrin structural domain begins with serine.

The 106-amino acid sequence motifs of spectrin have been suggested to fold into stable structural domains, consisting mostly of coiled coils of triple helices. With the advent of molecular biology and biophysical techniques, structural studies of these spectrin 106-amino acid structural domains became approachable. However, one of the difficulties in such an approach is determination of the correct phasing of the structural domains, which may or may not coincide with the phasing of the sequence motifs. Proper identification of the domain phasing is vital to the construction of stable spectrin domains for molecular studies. A previously published phasing shift for Drosophila alpha-spectrin indicated a downstream phase-shift of 26 amino acids for the structural domain (Winograd, E., Hume, D., and Branton, D. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10788-10791). Using this phase-shift, we prepared a recombinant spectrin peptide with the sequence from residue 49 to residue 155 of human erythrocyte alpha-spectrin and found this peptide to be unstable relative to other peptides that we prepared. Using several other recombinant alpha-spectrin peptides and following the protease digestion approach, we digested spectrin peptides with elastase and chymotrypsin and analyzed the amino acid sequence of the digestive products. We provide the first experimental evidence in identifying the first amino acid residue of the first spectrin domain in human erythrocyte alpha-spectrin as residue 52 (Ser).

Fourier transform infrared spectroscopic studies of the secondary structure of spectrin under different ionic strengths.

Spectrin, a highly dynamic skeletal membrane protein, plays an important role in maintaining the disk biconcave shape of the human erythrocyte. The sequence of spectrin is mostly composed of repeating segments of 106 amino acids which have been proposed to form unique structural domains. Electronic and vibrational circular dichroism and Fourier transform infrared (FTIR) spectroscopy were used as complementary techniques to study the secondary structure of spectrin. The amide I and II regions of the FTIR absorbance spectra were analyzed using partial least-squares analysis. The secondary structure of spectrin under physiological buffer conditions was estimated to be about 70% alpha-helix, 10% beta-sheet, and 20% other. We believe that this is the first detailed experimental evidence of significant beta-sheet content in spectrin secondary structure. The antiparallel beta-sheet SH3 domain in the center of the alpha-subunit in spectrin accounts for only about 1.5% of the total amino acid residues in the dimer. Hydrodynamic studies have shown spectrin to be sensitive to changes in ionic strength and to addition of denaturing agents. Our FTIR results showed that the secondary structure of spectrin treated with detergent or NaOH changed by 10-20%. The Stokes radii of the spectrin samples used for FTIR measurements were found to vary as a function of the ionic strength, but their secondary structures did not change as a function of ionic strength. These results indicate that while the overall hydrodynamic dimension of spectrin depends on the medium ionic strength, the secondary structure remains essentially constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipid vesicles promote human hemoglobin oxidation.

We have studied the heme oxidation kinetics of purified human hemoglobin (Hb) in the presence of lipid vesicles of dipalmitoyl phosphatidylcholine and bovine brain phosphatidylserine that exhibited minimal lipid peroxidation. We showed that the lipid vesicles enhanced Hb oxidation and that small unilamellar vesicles (SUVs) exerted a larger effect than large unilamellar vesicles (LUVs). We have determined pseudo first-order rate constants for the initial disappearance of oxygenated ferrous Hb (k0) and for the initial formation of several ferric Hb species (methemoglobin, hemichrome, and choleglobin) in the presence of SUVs and LUVs. k0 and other rate constants depended linearly on lipid-to-hemoglobin molar ratio (lipid/Hb), with k0SUV (h-1) = k0auto (h-1) + 3.7 x 10(-3) x lipid/Hb, and k0LUV (h-1) = k0auto (h-1) + 0.2 x 10(-3) x lipid/hb, where k0auto is the rate constant for Hb autoxidation in the absence of vesicles. Thus, in the absence of lipid peroxidation products, lipid vesicles themselves promote Hb oxidation by enhancing the rate of Hb oxidation. The enhanced oxidation was inhibited by catalase, but not by butylated hydroxytoluene. The rate constants were independent of Hb concentration, in the range of about 3.1 to 100 microM. We suggest that the lipid surface properties, including surface curvature, surface energy, and hydrophobicity, promote hemoglobin oxidation.

Dynamic light scattering investigations of human erythrocyte spectrin.

Dynamic light scattering measurements were performed on spectrin from human erythrocytes in 25 mM Tris buffer at pH 7.6 with 100 mM NaCl and 5 mM EDTA. Measurements were made on spectrin solutions prepared as dimers and tetramers over the temperature range from 23 to 41 degrees C, as a function of the square of the scattering vector (K2) over the range of 0.7 x 10(10) cm-2 less than or equal to K1 less than or equal to 20 x 10(10) cm-2. Analysis of the autocorrelation functions collected for these solutions revealed the presence of two predominant motional components over the entire range of K2. Plots of the diffusion coefficients (D20) of these components, with viscosity and temperature corrected to water at 20 degrees C, as a function of K2 indicated three rather distinct regions, flat regions at low and high K2 joined by a sloping intermediate region. At small K2 (less than or equal to 4 x 10(10) cm-2) the D20 values were (7.3 +/- 2.0) x 10(-8) cm2/s for the slow component and (20.3 +/- 2.0) x 10(-8) cm2/s for the fast component. At large K2 (greater than or equal to 10 x 10(10) cm-2) the values increased to (13.0 +/- 2.0) x 10(-8) cm2/s for the slow component and (39.4 +/- 2.0) x 10(-8) cm2/s for the fast component. In the intermediate K2 region, D20 is a linear function of K2 and appears as a transition between the low and high K2 regions.(ABSTRACT TRUNCATED AT 250 WORDS)