Parakeet Hemoglobin - Its Crystal Structure and Oxygen Affinity in Relation to Some Avian Hemoglobins.

BACKGROUND: "Avians" often show efficient oxygen management to meet the demands of their metabolism. Hemoglobin, a transporter protein consists of four non-covalently linked subunits contain haem binding hydrophobic pocket serves as a site of allosteric cooperativity. The physiology and anatomy of both mammals and avian are functionally different, in birds, the respiratory system formed by small air sacs that serve as tidal ventilation for the lungs and have no significant exchange across their cells. Parakeet (Psittacula krameri) a tropical and non-migrating species and it is easily adapted to living in disturbed habitat. The sequence analysis reveals that α and ß chain of parakeet hemoglobin highly similar grey lag goose and bar headed goose hemoglobin respectively. Thus it has been tempted us to study in to analyzing the sequence and structural comparison of this hemoglobin to find out the physiological capabilities of parakeet hemoglobin. OBJECTIVE: The structure determination studies of parakeet hemoglobin by X-ray diffraction. The sequence and structure are compared with goose, chicken and human Hb, emphasizing the role of amino acids in the subunit contacts that facilitate survival by low oxygen demand. METHODS: The Hb was purified and crystallized by hanging drop vapor diffusion method using poly ethylene glycol (PEG) 3350 and sodium phosphate buffer. X-ray diffracted data set was collected at 3Å resolution, the data was processed in Automar and molecular replacement, refinements, model building was carried out in CCP4i program package. The final refined model was deposited in protein data bank with accession id 2zfb. RESULTS: The tertiary structure of Parakeet Hb is compared with the met form of BHG Hb (1c40) and oxy form of GLG (1faw) and oxy form of human Hbs (1hho). Superimposing parakeet Hb α1ß1 subunit with 'R' state human Hb shows an r.m.s.d of 0.98 Å and for BHG and GLG Hb, the r.m.s.d shows 0.72 and 0.61 Å. The replacement of α115Asp in parakeet Hb as against the α115Glu in human Hb results in the movement of GH corners. The amino acid proline at α50 present only in Parakeet Hb and Chicken HbD and not present in any other avian family which includes human Hb. The residue α78Thr located in EF corner loop region, which slightly diverge when superimposing with human and BHG Hb and also replacement of α113Asn present only in Parakeet Hb placed near the FG helix corner. CONCLUSION: The present study describes the structure determination of parakeet hemoglobin and its structural features to understand its oxygen affinity characteristics. The crystals were obtained by buffered low-salt conditions, like those of chicken HbD, carbonmonoxy and cyanomet human Hb. The present study reveals several interesting and unique modifications in the finer aspects of the quaternary structure of parakeet Hb, which are involved in oxygen affinity characteristics and the α1ß1 subunit contacts. Crystallization of parakeet Hb with allosteric effectors like Inositol pentaphosphate may bring further understanding of the influence of physiological and environmental factors on the quaternary structure.

Purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies of catfish (Clarias magur) haemoglobin.

Haemoglobin is an interesting physiologically significant protein composed of specific functional prosthetic haem and globin moieties. In recent decades, there has been substantial interest in attempting to understand the structural basis and functional diversity of fish haemoglobins (Hbs). Towards this end, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies have been carried out on Clarias magur Hb. Crystals were grown by the hanging-drop vapour-diffusion method using PEG 2000 and NaCl as precipitants. The crystals belonged to the primitive monoclinic system P2, with unit-cell parameters a=98.35, b=56.63, c=112.88 Å, ß=100.22°; a complete data set was collected to a resolution of 2.4 Å. The Matthews coefficient of 2.42 Å3 Da(-1) for the crystal indicated the presence of two α2ß2 tetramers in the asymmetric unit.

Purification, crystallization and preliminary X-ray diffraction studies of parakeet (Psittacula krameri) haemoglobin.

Birds often show efficient oxygen management in order to meet the special demands of their metabolism. However, the structural studies of avian haemoglobins (Hbs) are inadequate for complete understanding of the mechanism involved. Towards this end, purification, crystallization and preliminary X-ray diffraction studies have been carried out for parakeet Hb. Parakeet Hb was crystallized as the met form in low-salt buffered conditions after extracting haemoglobin from crude blood by microcentrifugation and purifying the sample by column chromatography. Good-quality crystals were grown from 10% PEG 3350 and a crystal diffracted to about 2.8 A resolution. Preliminary diffraction data showed that the Hb crystal belonged to the monoclinic system (space group C2), with unit-cell parameters a = 110.68, b = 64.27, c = 56.40 A, beta = 109.35 degrees . Matthews volume analysis indicated that the crystals contained a half-tetramer in the asymmetric unit.

Studies of collagen in bone and dentin matrix of a Columbian mammoth (late Pleistocene) of central Utah.

A Columbian mammoth, Mammuthus columbi, was excavated at an elevation of 9000 feet in Huntington Canyon, Emery County, Utah. Radiocarbon dates on the skeleton indicated death approximately 11,200 years ago. The skeleton was removed from postglacial, Late Quaternary, lake sediments deposited as glacial runoff approximately 9500 years ago. The bones and teeth were especially well preserved in a saturated lake bed. After excavation the bones and teeth were preserved by controlled desiccation, without hardeners, over a period of 9 months. Microradiography, light and electron microscopy, medium and high angle X-ray diffraction, amino acid analysis and cyanogen bromide peptide mapping were undertaken to evaluate the packing, organization, and preservation of collagen in bone and dentin of this mammoth. Microradiography and light microscopy showed that the bone consisted of especially well preserved compact and trabecular bone, and electron microscopy of demineralized bone and tusk showed that the matrix consisted of lamellae of densely packed cylindrical collagen fibrils. Cell remnants with intact nuclei, with or without a nucleolus, as well as variable lengths of plasma membrane were occasionally present on the surface of bony trabecula. Remnants of odontoblast processes were present in some dentin tubules. High and low angle X-ray diffraction demonstrated that the demineralized matrix contained native collagen molecules and amino acid analysis showed that the composition was comparable to that of type I collagen. Cyanogen bromide peptide mapping indicated that the major peptides of type I collagen were present and had the same electrophoretic mobility as that of type I collagen of demineralized Asian elephant bone and rat tail tendon.(ABSTRACT TRUNCATED AT 250 WORDS)

X-ray diffraction studies on human tendon show age-related changes in collagen packing.

In this report, X-ray diffraction on native hydrated tendon is established as a monitor of human aging. X-ray diffraction patterns were recorded on toe extensor tendons of persons ranging from 1.6 years to 87 years old. All patterns show a set of 67 nm meridional reflections derived from the collagen fibril axial repeat, and the ratio of the intensities of the 16:17 orders showed a linear increase with age. The spacing of the equatorial maximum, which relates to the lateral packing of molecules in collagen fibrils, was also greater in older tendons. The observed changes in X-ray parameters follow those seen for rat-tail tendons subjected to in vitro non-enzymatic glycosylation. Age-related increases reported for the fluorescence of Maillard products and the concentration of the sugar-derived cross-link pentosidine are similar to the trends in X-ray parameters reported here. Our results support the cumulative nature of non-enzymatic glycosylation in connective tissues during the human lifespan and indicate that structural changes accompany the chemical alterations. The X-ray parameters show a large degree of scatter for ages older than 60 years, suggesting other complicating factors are present. Studies on a small number of diabetic tendons show small, but not significant, increases compared to age-matched controls.