Reexamination of the polymerization of pyridoxylated hemoglobin with glutaraldehyde

Pyridoxylated adult human hemoglobin (HbAo) was prepared using a one molar equivalent of pyridoxal 5-phosphate (PLP) per heme and reduced with either NaCNBH3 or NaBH4. A separate sample was pyridoxylated and passed through a mixed-bed ion exchange column without reduction. All three preparations had a P50 of 29 +/- 2 torr and a cooperativity of n = 2.4 +/- 0.1. These preparations, in both the oxy and deoxy forms, were then treated with 7 equivalents of glutaraldehyde per tetramer at pH 6.8 at 4 degrees C and at room temperature. The polymerization invariably reduced the P50 to 18 +/- 2 torr with Hill coefficients of less than 2. These solutions, with or without further reduction using NaCNBH3, all retained the PLP in differing amounts (2-3 moles/tetramer). Methemoglobin concentrations were increased during the polymerization reaction. The normal pyridoxylation procedure, using sodium borohydride reduction, resulted in a number of different molecular species. Polymerization with glutaraldehyde caused a further proliferation of molecular species that could not be separated by anion exchange chromatography or by isoelectric focusing. The extent of polymerization, estimated by gel exclusion chromatography and SDS polyacrylamide gel electrophoresis, was from 40 to 50%. Analysis of the reverse phase chromatograms, which separate the heme and the alpha- and beta-chains, showed extensive polymerization and distribution of the radioactively labeled PLP on the protein for all preparations. All of the polymerized and pyridoxylated samples were unstable, and showed different chromatographic patterns after storage at 4 degrees C for 1 month. Attempts to stabilize these preparations by further reduction with NaCNBH3 gave products with a lower P50 and lower cooperativity. When the reactions were conducted with a purified HbAo, heterogeneity was somewhat decreased compared to the normally used stroma-free hemoglobin, but a large number of molecular species were still formed.     

Characterization of tertiary interactions in a folded protein by NMR methods: studies of pH-induced structural changes in human growth hormone.

The pH-induced conformational changes in human growth hormone (hGH) have been studied, using a new quantitative NMR approach that combines 13C labeling of specific backbone carbonyl carbons with a complete spectral analysis of the corresponding 13C resonances. Thus, a complete analysis of the carbonyl resonances of the 26 Leu residues of hGH and their variation with pH provided detailed information about the equilibrium folding processes of the protein, including information about the kinetics of the folding. By combining this information with the pH dependence of readily identifiable 1H resonances, the pH-induced changes observed in the carbonyl carbon spectra can be associated with specific regions in the protein and can be ascribed to a series of localized adjustments in the tertiary structure, brought about by changes in the hydrogen bond interactions or electrostatic interactions between different residues in the globular folded protein. The preexchange lifetimes of these adjustments range from a fraction of a millisecond to a few milliseconds.     

Strontium Diffusion in Magnetron Sputtered Gadolinia‐Doped Ceria Thin Film Barrier Coatings for Solid Oxide Fuel Cells

Strontium (Sr) diffusion in magnetron sputtered gadolinia‐doped ceria (CGO) thin films is investigated. For this purpose, a model system consisting of a screen printed (La,Sr)(Co,Fe)O_3?δ (LSCF) layer, and thin films of CGO and yttria‐stabilized zirconia (YSZ) is prepared to simulate a solid oxide fuel cell. This setup allows observation of Sr diffusion by observing SrZrO₃ formation using X‐ray diffraction while annealing. Subsequent electron microscopy confirms the results. This approach presents a simple method for assessing the quality of CGO barriers without the need for a complete fuel cell test setup. CGO films with thicknesses ranging from 250 nm to 1.2 μm are tested at temperatures from 850 °C to 1000 °C which yields an in‐depth understanding of Sr diffusion through CGO thin films that may be of high scientific and technical interest for implementation of novel fuel cell materials. Sr is found to diffuse along column/grain boundaries in the CGO films but by modifying the film thickness and microstructure the breaking temperature of the barrier can be increased.     

Effects on Turkey Poults of Rations Containing Corn Invaded by Fusarium tricinctum (Cda.) Sny. & Hans

Consumption of an otherwise balanced ration containing 1% of corn invaded by Fusarium tricinctum isolate 2061-C resulted in the death of 13% of turkey poults within 35 days, in decreased feed efficiency and weight gain, and moderate development of bilateral necrotic lesions at angles of the mouth, especially in those that succumbed. Consumption of a ration with 2% of corn invaded by F. tricinctum resulted in death of 60 to 83% of the birds, in greatly reduced growth and feed efficiency in the survivors, and in development of severe mouth lesions. Consumption of rations containing 5, 10, and 20% of corn invaded by the fungus resulted in death of all birds in 5 to 15 days.     

Species Differentiation of DNA using Dot-Blots with Biotinylated or 32P-Labeled Genomic DNA Probe

Recombinam DNA technologies offer new methods of detecting animal diversity at various levels. DNA »fingerprinting« using cloned repetitive sequences can be used to distinguish between individuals within breeds and may thus be used for paternity testing (Jeffreys et al. 1985). The forensic value of evidence of this kind has given guarded approval by the UK Immigration Advisory Service in its annual report (Nature 1987). At the between-species level labeled total genomic DNA was used by Durnam et al. (1985) as probe in an in situ hybridization for speciesverification of chromosomal materials in human-rodent hybrid cell lines, and we recently described the use of 3H-labeled genomic pig DNA probe for detection af swine chromosomes in swine-hamster hybrid cell lines (Thomsen & Christensen 1986).