Purification and structure elucidation of Penicillium chrysogenum derived antifungal compound with potential anti-Candida property: in silico and in vitro evidence.

Following preliminary bioactivity testing, the fungal strain identified as Penicillium crysogenum was cultured in a modified Czapec Yeast Broth medium (CYB) for the production of antifungal compounds. Several chromatographic techniques including HPLC were used to purify the fungal metabolites from the crude extract. The mass determination of the purified compound was performed using Water's LCMS system while the structure of the compound was elucidated using 400 and 500 Varian NMR machines. The chemical name of the purified compound is (2 R, 4S) -2, 4-dimethyl-4-((E)-2-((3S, 4S)-2, 4, 5-trihydroxy-3-methoxy-4-phenyl-1, 2, 3, 4-tetrahydroquinolin-6-yl) vinyl) cyclohexanone with the chemical formula C26H31NO5 and exact mass of 437.2. Molecular docking predicted compound docking score with dihydrofolate reductase enzyme and lanosterol 14α-demethylase enzyme as -8.1 kcal/mol and -9.8 kcal/mol respectively. Further, the compounds showed stable binding mode with the enzymes and reported robust binding energies. After insilico analysis, the compound with mass 437 was tested for its antifungal potential in vitro against two pathogenic yeast species (i.e. Candida albicans and Candida glaberata) using the agar tube diffusion method. Using sterile di-methyl sulfoxide (DMSO) the compound was prepared in four dose concentrations (100, 250, 500, 1000 µg mL-1) and mixed with autoclaved semisolid Potato Dextrose Agar (PDA) medium in screw-capped test tubes labelled with the corresponding dose concentration. The fungal strains were inoculated on this medium and linear growth inhibition of the fungal strains was calculated using fluconazole as the control drug. The results from in vitro experiments were encouraging as at concentrations of 500 and 1000 µg mL-1 the compound inhibited the growth of C. albicans by 17% and 38% while 19% and 41% inhibition were recorded against C. glaberata. The compound showed antifungal activity in silico and in vitro against both the Candida species and can act as a potent antifungal candidate in the future upon further investigation.Communicated by Ramaswamy H. Sarma.

Aspergillus flavus originated pure compound as a potential antibacterial.

PROBLEM BACKGROUND: Penicillin was the first and most famous fungal secondary metabolite used as broad spectrum antibiotic that revolutionarised pharmaceutical research and also saved millions of lives. The over optimistic belief in 1967 that sufficient antibiotics had been discovered to defeat infectious diseases was quickly crashed with the appearance of multidrug resistant (MDR) bacteria in 1990s. This has posed a serious threat to mankind. Although scientists are making efforts to synthesize and discover new antibiotics there are not enough new drugs in pharmaceutical pipeline to beat the pace at which MDR bacteria are emerging. In view of this there is an urgent and serious medical need for new bioactive compounds to be discovered to treat infections caused by MDR pathogens. The present study is aimed to investigate the antibacterial potential of Aspergillus flavus originated compounds that may act as drug leads to treat future infections. METHODOLOGY: Among the 6 isolated fungal strains from the rhizosphere of Mentha piperetta, one was processed for isolation of secondary metabolites on the basis of preliminary antibacterial testing. Observation of morphological and microscopic features helped in identification of the fungal strain as Aspergillus flavus. Potato Dextrose Agar (PDA) medium was used for fungal growth while Czapec Yeast Broth (CYB) medium was used for production of fungal metabolites. Column chromatography technique was utilized for purification of compound from crude fungal extract and the mass of the compound was determined using Liquid Chromatography Mass Spectrometry (LCMS) method. Structure elucidation of the pure compound was performed using 500 Varian Nuclear Magnetic Resonance (NMR) machine. Docking was performed using Glide SP algorithm. Agar well diffusion method was used to determine the invitro antibacterial potential of the compound against two MDR bacterial strains i.e. Staphylococcus aureus and Proteus vulgaris. For this a total of 4 dose concentrations i.e. (100, 250, 500, 1000 µg mL- 1) of the compound were prepared and applied to bacterial strains on Mueller Hinton agar using tetracycline as control. RESULTS: The chemical name of the purified compound from A. flavus was determined as (2E)-3-[(3S, 4R)-8-hydroxy-3, 4-dimethyl-1-oxo-3, 4-dihydro-1H-2- benzopyran-7-yl] prop-2-enoic acid with the formula C14H14O5 and exact mass of 262.08. The in-Silico analysis showed that this compound has the potential to inhibit the binding pocket of S. aureus TyrRS (1JII) with docking score of - 8.67 Kcal mole- 1. The results obtained from invitro experiments were encouraging as at 1000 µg mL- 1 the compound showed 58.8% inhibition against S. aureus and 28% inhibition against P. vulgaris. CONCLUSIONS: The pure compound with formula C14H14O5 and exact mass of 262 exhibited antibacterial potential both insilico and invitro against both Gram negative and Gram positive bacteria. The compound was more active against S. aureus in comparison to P. vulgaris. From the obtained results it is concluded that this compound can be used as potent antibacterial candidate but further studies will be needed prior to its use as antibiotic.

Synthesis, characterization and antibacterial activity of simple ZnO and metal doped ZnO nanoparticles.

The objective of this study was to synthesize pure ZnO and metal doped ZnO nano-particles, determine its physical, chemical characteristics and antibacterial activity against selected bacterial strains. Pure ZnO was synthesized and metals including Manganese (Mn), Magnesium (Mg), Calcium (Ca), Copper (Cu) and Silver (Ag) were doped with ZnO to produce nanoparticles through co-precipitation method. X-ray diffraction (XRD), scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy were used for detection of synthesized nanoparticles, their crystalline structures, size and other chemical characteristics. An altered version of Kirby Bauer method of disk diffusion was used for determining the antibacterial activity of these nanoparticles. The XRD results showed that the average size of pure ZnO nanoparticles was 55.3 nm. While the size of metal doped ZnO particles were affected by dopant elements. Results of SEM indicated that these nanoparticles were roughly spherical, rod shape and fiber like rod shape with certain degree of aggregation. Antibacterial studies showed that all samples had the potential to inhibit the growth of selected bacterial strains; E. coli, S. choleraesuis, S. typhimurium, S. marcescens, B. subtilus and S. aureus. With 90µg/ml concentration ZnO nanostructures inhibited B.subtilus and silver doped Zinc nanoparticles suppressed growth of S. marcescens. Characterization and antibacterial study indicated the importance of these nanoparticles at industrial and pharmaceutical level.

Identification of risk factors for human immunodeficiency virus-1 infection in Khyber Pakhtunkhwa population: A case control study.

OBJECTIVES: The present study aims to identify the risk factors for Human Immunodeficiency Virus-1(HIV-1) infection in Khyber Pakhtunkhwa (KP) population by comparing HIV-antibody positive cases with HIV-antibody-negative controls. METHODS: The study was designed at the Family Care Centre (FCC), Hayatabad Medical Centre (HMC) Peshawar from February 2015 to December 2016. A total of 280 individuals were selected randomly for the study as cases and control. Data was collected on a structured questionnaire with informed oral consent. The collected data was analysed statistically using SPSS version 20. RESULTS: Out of 280 individuals, 56% were males, 44% were females, and 53.21% belonged to the urban areas. The literacy rate was 48.6%, and 75.4% were married. The statistical analysis of risk factors revealed the following factors as of significance value (p < 0.05). Family history of HIV (OR = 9.46), spouse status of HIV (OR=22.22), injection drug users (IDUs), migrants (OR=2.234), use of therapeutic injections (OR= 2.791), employment (OR=2.545), male gender (OR=2.35), tattooing (OR=7.667) and history of blood transfusion (OR= 2.69). CONCLUSION: The present study revealed spouse status of HIV, tattooing, migrants, IDUs, use of therapeutic injections, history of blood transfusion, male gender and employment as significant risk factors for HIV infection in the population of KP.

Herbicidal activity of pure compound isolated from rhizosphere inhabiting Aspergillus flavus.

In the quest for bioactive natural products of fungal origin, Aspergillus flavus was isolated from rhizosphere of Mentha piperita using Potato Dextrose Agar (PDA) and Czapec Yeast Broth (CYB) nutrient media for metabolites production. In total, three different metabolites were purified using HPLC/LCMS and the structures were established using 500 Varian NMR experiments. Further the isolated metabolites in different concentrations (10, 100, 1000 µg/mL) were tested for herbicidal activity using Completely Randomized design (CRD) against the seeds of Silybum marianum and Avena fatua which are major threats to wheat crop in Pakistan. Among the isolated metabolites, one compound was found active against the test weed species whose activity is reported in the present work. The chemical name of the compound is 2-(1, 4-dihydroxybutan-2-yl)-1, 3-dihydroxy-6, 8-dimethoxyanthracene-9, 10(4aH, 9aH)-dione with mass of 388. Results showed that all seeds germinated in control treatment; however, with the metabolite treated, the growth was retarded to different levels in all parts of the weeds. At a dose of 1000 µg/mL of the pure compound, 100% seeds of S. marianum and 60% seeds of A. fatua were inhibited. Interestingly, the pure compound exhibited less inhibition of 10% towards the seeds of common wheat (Triticum aestivum).

Synthesis of secondary metabolites by Cladosporium resinae (NRL-6437) under different growth media and chemical inducers and their pharmaceutical activity.

The role of different growth media and chemical enhancer on synthesis of secondary metabolites Cladosporium resinae (NRL-6437) was investigated for their in vitro biological activities. Cladosporium resinae (NRL-6437) were grown in various nutrient media (Czapeak-dox Broth (CB), Czapeak Yeast-extract Broth (CYB), Yeast Extract Sucrose (YES), Potato Dextrose Broth (PDB) and Czapeak-dox (supplemented with glucose and starch) Broth (CGSB) for the production of metabolites. Two chemical epigenetic modifiers (suberoyl-anilide hydroxamic acid (SAHA) and 5-azacytidine (5-AZA) were also used for the expression of silent genes for secondary metabolite production. Our results indicated that among different media, Czapeak yeast extract broth produced more secondary metabolites. Application of 15mM of both modifiers was effective for the expressions of silent genes resulting in an increased metabolites production. Secondary metabolites extracted in ethyl acetate and fractionized in n-Hexane were also tested for their biological activity. The secondary metabolites revealed varying degrees of growth inhibitions of the tested organisms. Similarly, these metabolites were also active against brine shrimps and Lemna.

Comparative studies on rabbit plasma lipid profile fed with Silybum marianum oil, sunflower oil and vegetable ghee.

Present work is aimed to compare the physicochemical characterization and biochemical effects of oil extracted from Silybum Marianum and Sunflower oil, collected from Peshawar (Pakistan). To investigate the comparative effects on the body weight, organ weight and lipid profile, the crude oil of Silybum marianum, edible sunflower oil and vegetable ghee were given to three groups of rabbits under study. Percent proximate composition and food consumption of all rabbits were determined which showed no significant statistical variation. There is no data available about Silybum marianum oil on animal model in literature. This study clearly revealed that oil from Silybum marianum significantly reduces plasma cholesterol level in rabbits. A threefold higher Triglyceride levels was observed in vegetable ghee feeding groups compared with the sunflower and Silybum marianum oil feeding groups. The crude oil of Silybum marianum was found to be safe in rabbits compared with sunflower oil and vegetable ghee. The results of these studies revealed most valuable information and also support the refining and purification to convert this non-edible oil to edible oil.

Evaluation of Non-Structural Protein-1(NS1) positive patients of 2013 dengue outbreak in Khyber Pakhtunkhwa, Pakistan.

BACKGROUND & OBJECTIVE: Dengue infection is an arthropod borne disease caused by Dengue virus in humans. Dengue virus infection has more potential to produce severe form of the disease with more severe symptoms. Proper diagnosis of dengue fever is very important for its safe management. The objective of this study was to evaluate the non structural protein-1 (NS1) positive parameter for identification of dengue fever by using ELISA from 2013 dengue outbreak in Khyber Pakhtunkhwa. METHODS: It was a cross sectional study conducted among 384 patients tested for dengue admitted to different hospitals of Khyber Pakhtunkhwa April to December 2013 with symptoms related to classical dengue fever. Written informed consent was taken from 100 NS1 positive diagnosed patients, and 3 to 5 ml blood sample was collected for confirmation through ELISA testing. ELISA test for dengue IgG and IgM was performed two time in order to confirm the dengue cases. Data was entered and analyzed by using SPSS version 16. RESULT: The study performed on 100 NS1 positive samples of patients, admitted to hospitals with symptoms related to classical dengue fever, indicated that after performing the IgM and IgG capture ELISA test only 76 samples were actually found positive for dengue. The rest of the 24 samples were found negative for both IgM and IgG capture ELISAs. The study also revealed that 90.8 % patients had primary dengue infection and 35.5% patients had secondary dengue infection. Most patients were between the age of 10-20 years (26%), among them19.7% were having primary dengue infection. Among 10-20 years of age 50% female patients were false dengue patients. CONCLUSION: About 24 % NSI protein positive samples were found negative for both IgM and IgG capture ELISAs showed that NS1protein positivity does not confirm actual dengue infection.

Comparative study of heavy metals in dried and fluid milk in Peshawar by atomic absorption spectrophotometry.

Various essential and toxic heavy metals (Ca, Mg, Cu, Zn, Fe, Mn, Pb, Cd, Cr, and Ni) contents in various types of dried (infant formula and powdered) and fluid (fresh and processed) cow milk were assessed by atomic absorption spectrophotometry. The milk samples were collected from local markets of different parts of Peshawar city, Pakistan. Heavy metal concentrations varied significantly depending upon the type of milk. The heavy metal concentrations in most of the samples were within normal and permissible ranges. It was observed that the samples contained considerable amounts of calcium, while magnesium levels were well above the required levels. The results also revealed that copper levels were slightly lower than the permissible limits. The concentration of zinc in dried milk samples was greater than the values for the liquid milk types. Infant milk formulae had higher iron levels as compared to other milk samples because of the added constituents. Significant differences were observed in the mean values of manganese and cadmium in different types of milk. The toxic metals were within the acceptable limits and did not show significant levels leading to toxicity.

Prevalence of zinc deficiency among rural women during childbearing age in Peshawar, Pakistan.

Zinc deficiency is a commonly reported health problem throughout the world. This cross sectional survey was conducted in rural Peshawar with an aim to estimate the prevalence of zinc deficiency in women of child bearing age and find its association with age, marital, pregnancy status and parity. Data was collected from 353 women age 15-45 years. EPI INFO version 6.04 was used for data analysis. Overall 98 (27.8 %) women were zinc deficient (<80 µg/dL) while 31 (8.8%) had severe zinc deficiency (<50µg/dL.). Mean zinc level was found to increase gradually with the increase in the age up to 40 years and then starts decreasing significantly beyond this age. A significant decrease (p<0.03) in zinc concentration was found in married as compared to unmarried women. Out of 31 female with severe zinc deficiency, 23 (74.2%) were pregnant. Pregnant women in second (OR (CI) 3.36 (1.52-7.44) p<0.0008) and third ((OR (CI) 3.73 (1.91-7.30) p<0.00002) trimester were 3.4 & 3.7 times, respectively more zinc deficient as compared to control women. Mean zinc levels were significantly lower in women having no children versus women with 1-5 numbers of children. This study concludes that severe zinc deficiency especially prevalent in pregnant women needs urgent correction through food supplementation.

Analysis of oxygen affinity in aquatic amphibian; homology modelling of the major haemoglobin component HbA1 from the African clawed frog (Xenopus laevis, Anura).

The homology model of major haemoglobin component HbA1 of the African Clawed Frog was predicted using the pigeon (Columba livia) haemoglobin as a template. The model was built with the help of MODELLER9v8. The models were evaluated with ProSA and PROCHECK. In X. laevis Gln38α is unable to form a hydrogen bond with ß97His or ß99Asp, which is responsible for the increase in oxygen affinity of the Xenopus HbA1. The hydrogen bond between α34Thr and ß124Pro, which stabilises the deoxy state of the haemoglobin, was absent in X. laevis. Hence it is predicted that the HbA1 component of X. laevis has higher oxygen affinity.

Abnormal Lipid levels as a risk factor of eclampsia, study conducted in tertiary care Hospitals of Khyber Pakhtunkhwa Province - Pakistan.

OBJECTIVE: To evaluate abnormal lipid metabolism as a risk factor of eclampsia in pregnant women. METHODS: This cross sectional study was conducted in three tertiary care hospitals of Peshawar. Serum total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), very low density lipoprotein cholesterol (VLDL-C), triglyceride (TG), apolipoprotein A1 (APO-A1), APO-B100, lipoprotein-a (Lpa) were measured in 110 women with eclampsia and compared with 90 healthy pregnant women. Mean lipid levels in cases and controls were compared using student's t test". RESULTS: Mean systolic/diastolic blood pressure, TC, TG, VLDL-C and Lpa levels were significantly higher (p<0.001) in patients compared to control women. Similarly TC: HDL-C, LDL-C: HDL-C and TG: HDL-C ratio in the patients group were significantly higher (p<0.001) and HDL-C: VLDL-C ratio was significantly lower (p<0.001) in the patients as compared to control group. Undesirable cholesterol were noted in 35.8% patients, HDL-C in 50.5%, borderline high concentration of LDL-C in 23.6%, high triglycerides levels in 73.2%, undesirable cholesterol ratio in 52.3% and undesirable LDL-C ratio were noted in 82.1% patients of eclampsia. CONCLUSION: Serum lipids were found significantly higher thus early assessment may be helpful in prevention of complications in the eclampsia patients.

Structural and functional features of Streptolysin O.

Streptolysin O, a 63 kDa exotoxin coded by slo gene, is a well-characterised thiol-activated cytolysin, which damages cholesterol-containing membranes resulting in disruption and lysis of the target cell. On the basis of homology model and secondary structure analysis, the toxin has four domains of which domain 4 is of particular importance and is directly linked to domain 2 by a glycine linker and remained consistent in initial membrane recognition. Domain 4 reduces the hydrophobic ratio when compared with its template, which would affect the activity of the toxin at low pH.

Structural modeling studies of aldehyde dehydrogenase X: insights into key interactions in the tetrameric assembly of the isoenzyme.

Human mitochondrial aldehyde dehydrogenase is a member of superfamily of multisubunit enzymes, catalyzing the conversion of a broad range of aldehydes to corresponding acids via the NAD (P) (+)-dependent irreversible reaction. They play an important role in the detoxification of acetaldehyde, in the development of alcohol sensitivity and human alcohol-related disorders. The study aimed to understand the role of conserved residues by comparing similarities and differences between the two isoenzymes. A 3D model of the human ALDHX is constructed by molecular modeling based on the crystal structure of human ALDH2 by using MODELLER (8V1) program. Assessment of reliability of the 3D model is carried out by the programs PROCHECK and PROSAII. The ALDHX fold is similar to the previously described ALDH structures. Sequence and structural analyses have highlighted a close structural and functional relationship between the two isoenzymes of human origin. The interfacial residues that are involved in crucial interactions across the interface stabilize the dimer-tetramer interface in the enzyme. Stability factors like salt bonds and hydrogen bonds aid and maintain the tetrameric assembly of the enzyme.

Structural bioinformatics of Vibrio cholerae aminopeptidase A (PepA) monomer.

Aminopeptidase A (PepA) is a metalloexopeptidase found in Vibrio cholerae .It functions as a transcriptional repressor in regulatory cascade that controls virulence gene expression in V. cholerae. It is involved in protein degradation and in the metabolism of biologically active peptides. We proposed a 3D model of PepA based upon the crystal structure of PepA from Escherichia coli (E. coli) with an intention to evaluate the active site of the enzyme and to predict the properties of this enzyme, study of its 3D structure will help in understanding its role in DNA binding.

Homology modeling of hemagglutinin/protease [HA/P (vibriolysin)] from Vibrio cholerae: sequence comparision, residue interactions and molecular mechanism.

Vibrio cholerae produces a zinc-containing and calcium-stabilized soluble hemagglutinin/protease, which has been earlier shown to have the ability to cleave several physiologically important substrates including mucin, fibronectin and lactoferin. This study presents homology modeling of hemagglutinin/protease (vibriolysin) from Vibrio cholerae in the presence of inhibitor HPI [N-(1-carboxy-3-phenylpropyl)-phenylalanyl-alpha-aspargine]. The 3D structure was predicted based on its sequence homology with Pseudomonas aeruginosa elastase (PAE). Comparison of the 3D structures of PAE and HA/P reveals a remarkable similarity having a conserved alpha + beta domain. The inhibitor shows similar binding features as seen in other metalloproteases of M4 peptidase family. The study also highlights the key catalytic residues as well as the residues at the S1 and S1' binding sub-sites. The similarities between the two proteins provide support for the hypothesis that the two enzymes have similar three-dimensional structures and a common mechanism of action. The fact that both enzymes are secreted as zinc-containing proteases, led us to further hypothesize that they may play similar role in pathogenesis.

Low oxygen affinity in reptilian hemoglobin D: prediction of residue interactions in Geochelone carbonaria HbD by homology modeling.

The homology model of hemoglobin D from Geochelone carbonaria, the red-footed tortoise was predicted using the 3D structure coordinates of Geochelone gigantea hemoglobin D as the template. The model was built using the program, MODELLER (8v1) and evaluated with PROCHECK and PROSA. The present study features an in-depth analysis of the 3D model and its conformational changes brought about with variations in its environment. These structural changes are correlated with its ability to adapt to different environmental constraints enabling the organism to better suit to its natural habitat. The model shows additional contacts between amino acid pairs of alpha-119 and beta-55, alpha-35 and beta-124, alpha-103 and beta-112, alpha-115 and beta-116, alpha-120 and beta-52, alpha-120 and beta-55, alpha-36 and beta-127 which are not found in human hemoglobin. It is predicted that these contacts may result in T-state stabilization thus lowering oxygen affinity. Furthermore, decrease in the interaction of phosphate heteroatoms with the amino acid residues of G. carbonaria Hb was also predicted in this study.

Electrolytic recovery of chromium salts from tannery wastewater.

Tannery wastewater collected from a local leather industry in Peshawar, Pakistan was subjected to DC electrolysis in a simple cell having two static sheet electrodes and stirring assembly after proper dilution and adjustment to desired conditions. One percent HNO(3) and 1% NaHCO(3) were used as electrolytes and pH adjusters. The latter salt also worked as sodium source for anodic deposition of Na(2)Cr(2)O(7). Various combinations of electrodes were tested and conditions optimized for best electrode couple with increased recovery and removal of chromium in the form of Cr(OH)(3) and/or Na(2)Cr(2)O(7) at cathode and anode, respectively. The recovery of 99% chromium was achieved after 2h electrolysis at a cell potential of 1.0 V, pH 5.0 and stirring rate of 500 rpm using Pb sheet as anode and Cu sheet as cathode. The most interesting and novel finding of this work was the recovery of the mentioned salt(s) alone at Cu cathode or Pb anode or collectively at both electrodes by proper control of pH. Such treatment not only minimizes the environmental water pollution, but results in the formation of useful products employed for recycling purpose in tannery or other related industry to make the process economical.

Molecular mechanism of high altitude respiration: primary structure of a minor hemoglobin component from Tufted duck (Aythya fuligula, Anseriformes).

Avian hemoglobins have attracted much attention in view of the unique oxygen transport characteristics. The present study describes the primary structure of minor hemoglobin component HbD from Tufted duck (Aythya fuligula), a migratory bird seen in Pakistan during the winter season. Separation of the polypeptide subunits was achieved by ion exchange chromatography in the presence of 8M urea. Molecular masses of the intact protein as well as peptides obtained from chemical and enzymatic cleavages were determined by electrospray ionization mass spectrometry. The sequence was studied by automatic Edman degradation of the native chains and their tryptic/hydrolytic fragments in a gas-phase sequencer. Comparison of the hemoglobin sequence with the corresponding sequences of Anseriform representatives and other avian species shows residues like alpha(D)23 Asp, alpha(D)120 Asp as being specific to Tufted duck. The three-dimensional structure analyzed with the protein structure modeling package, WHAT IF, using the crystal structure coordinates of chicken hemoglobin (PDB code=1hbr) shows alpha(D)34 Val, alpha(D)38 Gln, and alpha(D)94 Asp as possible mediators offering alternate pathway for oxygen uptake and release thereby leading to distinct hypoxia tolerance in the Tufted ducks. Results are discussed with reference to function and evolution in the Anseriform representatives.

Molecular basis of bird respiration: primary hemoglobin structure component from Tufted duck (Aythya fuligula, Anseriformes)--role of alpha99Arg in formation of a complex salt bridge network.

The primary structure of the major hemoglobin component, HbA (alpha(A)- and beta-chain), from Tufted duck (Aythya fuligula) is presented. The separation of the globin subunits was achieved by ion exchange chromatography on CM-cellulose in 8 M urea. The amino acid sequence was determined by automatic Edman degradation of native chains as well as tryptic and hydrolytic peptides in a gas-phase sequencer. The automated homology model was generated by the protein structure modeling package WHAT IF using the crystal structure coordinates of Bar-headed goose hemoglobin. The 3D structure prediction enables alpha99Arg and beta101Glu to emerge as a new intersubunit contact site not found in the hemoglobin structure of any other species. alpha99Arg forms a complex salt bridge network involving alpha99Arg-beta101Glu-beta104Arg-beta108Asp. Also the substitution at alpha34 --> Ile, alpha38 --> Gln and beta55 --> Leu serves to stabilize the oxy-structure, leading to higher oxygen affinity.