Microvascular changes on nailfold capillaroscopy in acute stage of Kawasaki disease: a new diagnostic paradigm for an enigmatic condition.

OBJECTIVES: Kawasaki disease (KD) is a medium vessel vasculitis with a predilection to involve coronary arteries. However, there is a paucity of literature on microvascular changes in patients with KD. METHODS: Children diagnosed with KD based on American Heart Association guidelines 2017 were enrolled prospectively. Demographic details and echocardiographic changes in coronaries were recorded. Nailfold capillaries were assessed using Optilia Video capillaroscopy and data were analysed using Optilia Optiflix Capillaroscopy software at acute (prior to IVIG administration) and subacute/convalescent phase. RESULTS: We enrolled 32 children with KD (17 boys) with a median age of 3 years. Nailfold capillaroscopy (NFC) was performed in 32 patients in the acute phase (compared with 32 controls) and in 17 during the subacute/convalescent phase at a median follow-up of 15 (15-90) days after IVIG treatment. The following findings were seen in NFC in the acute phase of KD: reduced capillary density (n = 12, 38.6%), dilated capillaries (n = 3, 9.3%), ramifications (n = 3, 9.3%) and capillary haemorrhages (n = 2, 6.2%). Capillary density was reduced significantly in the acute phase of KD (38.6%) as compared with the subacute/convalescent phase (25.4%) (P-value <0.001) and controls (0%) (P-value = 0.03). We observed no correlation between coronary artery involvement and mean capillary density (P = 0.870). CONCLUSION: Results show that patients with KD have significant nailfold capillary changes in the acute phase. These findings may provide a new diagnostic paradigm for KD and a window to predict coronary artery abnormalities.

Pixel Grafting: A Novel Skin Graft Expansion Technique.

Skin grafting is the transplantation of skin, a routinely performed procedure to cover the loss of skin. Skin is the largest organ of the body, which falls short of availability in extensive injuries, especially burns. In such a situation, pixel grafting, a novel expansion technique helps to cover a large area with less skin harvest. The objective of the study was to test fast, minimally invasive, easy to use minced split-thickness skin graft to cover large wounds and to reflect on the advantages of pixel graft. It is a pilot study of patients admitted with severe burns. We conclude that with this technique of pixel or minced grafting, large areas can be grafted with minimal donor-site requirement, and the techniques of preparation provide adequate size graft for pixel grafting.

Monitoring of Chemical Markers in Extraction of Traditional Medicinal Plants (Piper nigrum, Curcuma longa) Using In Situ ReactIR.

BACKGROUND: The fingerprinting and quantification of marker compounds from medicinal plants is a domain of the herbal industry for quality/quantity control parameters. OBJECTIVE: The main objective of this study is the application of the in situ ReactIR technique for measuring the concentration of different components during the extraction process of different medicinal plants. METHOD: In this study we have performed the extraction of two-marker compounds, viz. piperine from Piper nigrum and curcumin from Curcuma longa plants, using various solvents (dichloromethane and methanol). The progress of extraction was monitored using an in situ Fourier transform infrared (FTIR) probe instrument and an automated reactor. RESULTS: In this communication, using the in situ ReactIR technique we developed a method which demonstrates the relative quantification of marker analytes, optimizes extraction time and type of solvents to be used for different analytes during the extraction process. CONCLUSIONS: To the best of our knowledge, this is the first report of relative quantification and structural information of marker compounds during the process of extraction using in situ FTIR. HIGHLIGHTS: The present study highlights the real-time monitoring, in situ quantification, and structural information of marker compounds during the process of extraction of medicinal plants using in situ FTIR.

Innovative Cost-Effective Application of Donut Dressing in Skin Grafting.

One of the measures for the successful take of a skin graft is the prevention of friction, especially in cases of pressure ulcers in patients with head injury leading to altered sensorium. With existing measures such as the use of a pressure-relieving bed, frequent change of position, etc graft loss is common. Some additional measures are required. This study highlights the role of a donut-shaped ring dressing to protect the skin graft from friction.

Evaluation of platelet-rich fibrin and tricalcium phosphate bone graft in bone fill of intrabony defects using cone-beam computed tomography: A randomized clinical trial.

BACKGROUND: Platelet-rich fibrin (PRF) is the second-generation platelet concentrate first described by Choukron et al. It incorporates leukocytes, platelets, and growth factors within dense fibrin matrix, can be used in periodontal regeneration alone or in combination with bone grafts. AIM: This study assesses bone fill in intrabony defects, following the use of ß tricalcium phosphate (TCP) bone graft with and without PRF. MATERIALS AND METHODS: Thirty sites with intrabony defects in periodontitis patients were selected, randomly allotted into three groups: Group A open flap debridement (OFD), Group B OFD with ß TCP with PRF, and Group C ß TCP. Clinical parameters such as plaque index, gingival index, sulcus bleeding index, and PPD recorded at baseline and 6 months. Radiographic parameters include cementoenamel junction (CEJ) to base of defect, CEJ to alveolar crest, depth of defect, and bone fill assessed using the cone-beam computed tomography (CBCT). The comparison between the test group and control group in terms of clinical and radiographical parameters was assessed using the independent sample t-test. RESULTS: Significant reduction in probing depth measurements, defect fill observed in both ß TCP with PRF and ß TCP alone groups compared to OFD. However, intergroup comparison assessed using the independent sample t-test found to be statistically nonsignificant (P < 0.05 is considered significant). CONCLUSION: All three treatment strategies resulted in significant reduction in probing depth and bone fill at 6 months. Bone fill achieved in ß TCP with PRF was more compared to ß TCP alone and OFD at 6 months follow-up. CBCT can be accurately used to assess the morphology of intrabony defect and also in evaluating bone fill.

Acute undifferentiated febrile illness: Protocol in emergency department.

Fever accounts for around 15% of emergency visits in elderly age group and around 5% in adults. The spectrum of etiologies ranges from non-infectious to infectious etiologies. There are very few studies done in the past highlighting the approach of patients with acute febrile illness without any localizing signs and symptoms. OBJECTIVES: The aim of the study was to formulate a targeted approach for evaluation and treatment of patients with acute undifferentiated febrile illness without evidence of localizing symptoms and signs. The secondary objective was to study the etiology and final outcome of patients with acute undifferentiated febrile illness. MATERIALS AND METHODS: A protocol was devised for patients aged more than 18 years, who presented in emergency department with complaints of fever without localizing symptoms or signs of sepsis over a period of 6 months from April 2018 to September 2018. Patient's data were collected retrospectively from the hospital record section. RESULTS: A total of 212 patients of undifferentiated acute febrile illness were enrolled in the study. Maximum number of patients [n = 69 (32.5%)], presented on second day of illness. All the patients presenting within 1 or 2 days of fever experienced defervescence. Out of these 69 patients, 35 (36.4%) were investigated of which in 29 (82.2%) investigations were not found to be useful; 75 (78.1%) patients with 1 or 2 days history of fever improved without investigations. Surprisingly, 54 patients (72%) with 1 or 2 days' history of acute febrile illness experienced defervescence without the need of antibiotics. CONCLUSION: There is an urgent need to devise a standardized protocol for diagnosis and treatment of patients with acute undifferentiated febrile illness in order to avoid unnecessary investigations and antimicrobial use.

Cadaveric Bilateral Proximal Forearm Allotransplantation.

Until, sometime ago, microsurgery meant mainly covering a defect or replanting severed parts back to where they belong. Now, restoration of original function and aesthetic consideration is a must in planning reconstructive procedure. Hand transplant combines hand surgery and microsurgery with complex multidisciplinary care.At the anniversary of our first cadaveric bilateral proximal forearm transplantation done in the country's government institute, we would like to share our experience in performing the surgery, outcomes so far, complications, and lessons learned, to contribute to the growing knowledge of vascularized composite allotransplant.

Effectiveness of Fibrin Glue in Adherence of Skin Graft.

BACKGROUND: Graft fixation is important for graft take. Fibrin glue has been proposed as an ideal material, because of its human origin and it provides firm adhesion in seconds or minutes. OBJECTIVE: To evaluate the efficiency of fibrin glue, in increasing the take of skin graft. Assessment includes surgical time taken for graft fixation, haematoma/seroma formation, engraftment and wound closure by day 14. METHODS: The study is an observational prospective study conducted in the Department of Plastic Surgery, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, from January 2016 to June 2016. Sixteen patients who underwent split skin grafting were assessed during the study period. Fibrin glue was used on the recipient bed before grafting. RESULTS: Better haemostasis and graft adhesion, with a significant reduction of surgical time, were noted. CONCLUSION: The safety profile of fibrin glue was excellent as indicated by the lack of any related serious adverse experiences. These findings demonstrate that it is safe and effective for attachment of skin grafts, with outcomes at least as good as conventional methods.

Role of RanBP9 on amyloidogenic processing of APP and synaptic protein levels in the mouse brain.

We previously reported that RanBP9 binds low-density lipoprotein receptor-related protein (LRP), amyloid precursor protein (APP), and BACE1 and robustly increased Aß generation in a variety of cell lines and primary neuronal cultures. To confirm the physiological/ pathological significance of this phenotype in vivo, we successfully generated transgenic mice overexpressing RanBP9 as well as RanBP9-null mice. Here we show that RanBP9 overexpression resulted in >2-fold increase in Aß40 levels as early as 4 mo of age. A sustained increase in Aß40 levels was seen at 12 mo of age in both CHAPS-soluble and formic acid (FA)-soluble brain fractions. In addition, Aß42 levels were also significantly increased in FA-soluble fractions at 12 mo of age. More important, increased Aß levels were translated to increased deposition of amyloid plaques. In addition, RanBP9 overexpression significantly decreased the levels of synaptophysin and PSD-95 proteins. Conversely, RanBP9-null mice showed increased levels of synaptophysin, PSD-95, and drebrin A protein levels. Given that loss of synapses is the best pathological correlate of cognitive deficits in Alzheimer's disease (AD), increased Aß levels by RanBP9 observed in the present study provides compelling evidence that RanBP9 may indeed play a key role in the etiology of AD. If so, RanBP9 provides a great opportunity to develop novel therapy for AD.

Optimized mixture of hops rho iso-alpha acids-rich extract and acacia proanthocyanidins-rich extract reduces insulin resistance in 3T3-L1 adipocytes and improves glucose and insulin control in db/db mice

Rho iso-alpha acids-rich extract (RIAA) from Humulus lupulus (hops) and proanthocyanidins-rich extracts (PAC) from Acacia nilotica exert anti-inflammatory and anti-diabetic activity in vitro and in vivo. We hypothesized that a combination of these two extracts would exert enhanced effects in vitro on inflammatory markers and insulin signaling, and on nonfasting glucose and insulin in db/db mice. Over 49 tested combinations, RIAA:PAC at 5:1 (6.25 microg/mL) exhibited the greatest reductions in TNFalpha-stimulated lipolysis and IL-6 release in 3T3-L1 adipocytes, comparable to 5 microg/mL troglitazone. Pretreatment of 3T3-L1 adipocytes with this combination (5 microg/mL) also led to a 3-fold increase in insulin-stimulated glucose uptake that was comparable to 5 microg/mL pioglitazone or 901 microg/mL aspirin. Finally, db/db mice fed with RIAA:PAC at 5:1 (100 mg/kg) for 7 days resulted in 22% decrease in nonfasting glucose and 19% decrease in insulin that was comparable to 0.5 mg/kg rosiglitazone and better than 100 mg/kg metformin. RIAA:PAC mixture may have the potential to be an alternative when conventional therapy is undesirable or ineffective, and future research exploring its long-term clinical application is warranted.

Design and engineering of an O(2) transport protein.

The principles of natural protein engineering are obscured by overlapping functions and complexity accumulated through natural selection and evolution. Completely artificial proteins offer a clean slate on which to define and test these protein engineering principles, while recreating and extending natural functions. Here we introduce this method with the design of an oxygen transport protein, akin to human neuroglobin. Beginning with a simple and unnatural helix-forming sequence with just three different amino acids, we assembled a four-helix bundle, positioned histidines to bis-histidine ligate haems, and exploited helical rotation and glutamate burial on haem binding to introduce distal histidine strain and facilitate O(2) binding. For stable oxygen binding without haem oxidation, water is excluded by simple packing of the protein interior and loops that reduce helical-interface mobility. O(2) affinities and exchange timescales match natural globins with distal histidines, with the remarkable exception that O(2) binds tighter than CO.

Structure and phospholipase function of peroxiredoxin 6: identification of the catalytic triad and its role in phospholipid substrate binding.

Peroxiredoxin 6 (Prdx6) is a bifunctional protein with glutathione peroxidase and phospholipase A(2) (PLA(2)) activities, and it alone among mammalian peroxiredoxins can hydrolyze phospholipids. After identifying a potential catalytic triad (S32, H26, D140) from the crystal structure, site-specific mutations were used to evaluate the role of these residues in protein structure and function. The S32A mutation increased Prdx6 alpha-helical content, whereas secondary structure was unchanged by mutation to H26A and D140A. Lipid binding by wild-type Prdx6 to negatively charged unilamellar liposomes showed an apparent rate constant of 11.2 x 10(6) M(-1) s(-1) and a dissociation constant of 0.36 microM. Both binding and PLA(2) activity were abolished in S32A and H26A; in D140A, activity was abolished but binding was unaffected. Overoxidation of the peroxidatic C47 had no effect on lipid binding or PLA(2) activity. Fluorescence resonance energy transfer from endogenous tryptophanyls to lipid probes showed binding of the phospholipid polar head in close proximity to S32. Thus, H26 is a site for interfacial binding to the liposomal surface, S32 has a key role in maintaining Prdx6 structure and for phospholipid substrate binding, and D140 is involved in catalysis. This putative catalytic triad plays an essential role for interactions of Prdx6 with phospholipid substrate to optimize the protein-substrate complex for hydrolysis.

Inhibition of hemoglobin S polymerization in vitro by a novel 15-mer EF-helix beta73 histidine-containing peptide.

Our mutational studies on Hb S showed that the Hb S beta73His variant (beta6Val and beta73His) promoted polymerization, while Hb S beta73Leu (beta6Val and beta73Leu) inhibited polymerization. On the basis of these results, we speculated that EF-helix peptides containing beta73His interact with beta4Thr in Hb S and compete with Hb S, resulting in inhibition of Hb S polymerization. We, therefore, studied inhibitory effects of 15-, 11-, 7-, and 3-mer EF-helix peptides containing beta73His on Hb S polymerization. The delay time prior to Hb S polymerization increased only in the presence of the 15-mer His peptide; the higher the amount, the longer the delay time. DIC image analysis also showed that the fiber elongation rate for Hb S polymers decreased with increasing concentration of the 15-mer His peptide. In contrast, the same 15-mer peptide containing beta73Leu instead of His and peptides shorter than 11 amino acids containing beta73His including His alone showed little effect on the kinetics of polymerization and elongation of polymers. Analysis by protein-chip arrays showed that only the 15-mer beta73His peptide interacted with Hb S. CD spectra of the 15-mer beta73His peptide did not show a specific helical structure; however, computer docking analysis suggested a lower energy for interaction of Hb S with the 15-mer beta73His peptide compared to peptides containing other amino acids at this position. These results suggest that the 15-mer beta73His peptide interacts with Hb S via the beta4Thr in the betaS-globin chain in Hb S. This interaction may influence hydrogen bond interaction between beta73Asp and beta4Thr in Hb S polymers and interfere in hydrophobic interactions of beta6Val, leading to inhibition of Hb S polymerization.

Light induces an increase in the pH of and a decrease in the ammonia concentration in the extrapallial fluid of the giant clam Tridacna squamosa.

The objective of this study was to examine whether 12 h of light exposure would lead to an increase in the pH of and a decrease in the concentration of total ammonia in the extrapallial fluid of the giant clam Tridacna squamosa. We also aimed to elucidate indirectly whether movements of ammonia and/or protons (H(+)) occurred between the extrapallial fluid and the outer mantle epithelium. The pH of the extrapallial fluid of T. squamosa exposed to 12 h of light was significantly higher than that of clams exposed to 12 h of darkness. Conversely, the total ammonia concentration in the extrapallial fluid of the former was significantly lower than that of the latter. In addition, the glutamine content in the mantle adjacent to the extrapallial fluid of clams exposed to 12 h of light was significantly greater than that of clams exposed to 12 h of darkness. These results suggest that in the extrapallial fluid of T. squamosa exposed to light, NH(3) combined with H(+) as NH(+)(4) and that NH(+)(4) was transported into the mantle and used as a substrate for glutamine formation. Injection of NH(4)Cl into the extrapallial fluid led to an instantaneous increase in the total ammonia concentration therein, but the total ammonia concentration decreased subsequently and returned to the control value within 1 h. This is in support of the proposition that NH(+)(4) could be transported from the extrapallial fluid to the mantle. Injection of HCl into the extrapallial fluid led to an instantaneous decrease in the pH of the extrapallial fluid. However, there was a significant increase in pH within 1 h in light or darkness, achieving a partial recovery toward the control pH value. The increase in pH within this 1-h period in light or darkness was accompanied by a significant decrease in the total ammonia concentration in the extrapallial fluid, which supports the proposition that H(+) could be transported in combination with NH(3) as NH(+)(4). Therefore, our results prompt a reexamination of the previous proposition that the removal of H(+) by NH(3) can facilitate calcification in molluscs in general and an investigation of the relationship between H(+) removal through NH(+)(4) transport and light-enhanced calcification in T. squamosa.

Sevoflurane-induced structural changes in a four-alpha-helix bundle protein.

The mechanisms whereby volatile general anesthetics reversibly alter protein function in the central nervous system remain obscure. Using three different spectroscopic approaches, evidence is presented that binding of the modern general anesthetic sevoflurane to the hydrophobic core of a model four-alpha-helix bundle protein results in structural changes. Aromatic residues in the hydrophobic core reorient into new environments upon anesthetic binding, and the protein as a whole becomes less dynamic and exhibits structural tightening. Comparable structural changes in the predicted in vivo protein targets, such as the gamma-aminobutyric acid type A receptor and the N-methyl-D-aspartate receptor, may underlie some, or all, of the behavioral effects of these widely used clinical agents.

Expression and characterization of a four-alpha-helix bundle protein that binds the volatile general anesthetic halothane.

The structural features of volatile anesthetic binding sites on proteins are being investigated with the use of a defined model system consisting of a four-alpha-helix bundle scaffold with a hydrophobic core. The current study describes the bacterial expression, purification, and initial characterization of the four-alpha-helix bundle (Aalpha(2)-L1M/L38M)(2). The alpha-helical content and stability of the expressed protein are comparable to that of the chemically synthesized four-alpha-helix bundle (Aalpha(2)-L38M)(2) reported earlier. The affinity for binding halothane is somewhat improved with a K(d) = 120 +/- 20 microM as determined by W15 fluorescence quenching, attributed to the L1M substitution. Near-UV circular dichroism spectroscopy demonstrated that halothane binding changes the orientation of the aromatic residues in the four-alpha-helix bundle. Nuclear magnetic resonance experiments reveal that halothane binding results in narrowing of the peaks in the amide region of the one-dimensional proton spectrum, indicating that bound anesthetic limits protein dynamics. This expressed protein should prove to be amenable to nuclear magnetic resonance structural studies on the anesthetic complexes, because of its relatively small size (124 residues) and the high affinities for binding volatile anesthetics. Such studies will provide much needed insight into how volatile anesthetics interact with biological macromolecules and will provide guidelines regarding the general architecture of binding sites on central nervous system proteins.

Aldolases a and C are ribonucleolytic components of a neuronal complex that regulates the stability of the light-neurofilament mRNA.

A 68 nucleotide segment of the light neurofilament (NF-L) mRNA, spanning the translation termination signal, participates in regulating the stability of the transcript in vivo. Aldolases A and C, but not B, interact specifically with this segment of the transcript in vitro. Aldolases A and C are glycolytic enzymes expressed in neural cells, and their mRNA binding activity represents a novel function of these isozymes. This unsuspected new activity was first uncovered by Northwestern blotting of a brainstem/spinal cord cDNA library. It was confirmed by two-dimensional fractionation of mouse brain cytosol followed by Northwestern hybridization and protein sequencing. Both neuronal aldolases interact specifically with the NF-L but not the heavy neurofilament mRNA, and their binding to the transcript excludes the poly(A)-binding protein (PABP) from the complex. Constitutive ectopic expression of aldolases A and C accelerates the decay of a neurofilament transgene (NF-L) driven by a tetracycline inducible system. In contrast, mutant transgenes lacking mRNA sequence for aldolase binding are stabilized. Our findings strongly suggest that aldolases A and C are regulatory components of a light neurofilament mRNA complex that modulates the stability of NF-L mRNA. This modulation likely involves endonucleolytic cleavage and a competing interaction with the PABP. Interactions of aldolases A and C in NF-L expression may be linked to regulatory pathways that maintain the highly asymmetrical form and function of large neurons.

Hormonal influence on amylase gene expression during Seabass (Lates calcarifer) larval development.

alpha-Amylase gene expression was detected in newly hatched seabass (Lates calcarifer) larvae and peaked at around first feeding. This suggests a greater importance of carbohydrates during early larval development than might be expected for carnivorous fishes. In vivo cortisol and triiodothyronine (T(3)) treatment of seabass larvae upregulated alpha-amylase gene expression. The identification of a functional glucocorticoid-response element (GRE) on the amylase gene promoter indicates that cortisol (glucocorticoid) stimulation of amylase gene expression is direct via GRE. However no TRE (thyroid-response element) was found on the amylase gene and its promoter, and various concentrations of T(3) (1nM-10microM) also did not induce alpha-amylase gene promoter activity in rat AR42-J cells transfected with the promoter construct, unlike dexamethasone treatment. This suggests that T(3) stimulation of amylase gene expression in vivo was indirect, probably secondary to its promotion of one or more developmental processes.

Characterization of the seabass pancreatic alpha-amylase gene and promoter.

Seabass (Lates calcarifer) pancreatic alpha-amylase gene was cloned and characterized. The alpha-amylase cDNA has 1620 bp and the deduced polypeptide has 522 amino acids. Southern blot indicated that there are two gene copies in the seabass genome. Sequence analysis showed that except for the loss of an intron in seabass, the coding region and the exon/intron boundaries are highly homologous to those of mammalian amylases. However, the promoter regions are distinctively divergent. To investigate the seabass amylase promoter, a series of deletion mutants was generated and fused to the luciferase reporter gene, followed by studies of their functional activity in rat AR42J cell line. Besides identifying several potential regulatory elements that have been previously identified in the human and mouse pancreatic amylase promoter, we have identified a glucocorticoid response element (GRE). However, while the human and mouse pancreatic amylase promoters are highly homologous between nucleotide -160 and transcription start site where GRE is located, the 5' promoter deletion mutants revealed that the GRE of the seabass amylase promoter was located far upstream -947 to -776 bp of the promoter. Site-directed mutagenesis of the putative GRE and electrophoretic mobility shift assays (EMSA) confirmed that this region was responsible for dexamethasone induction. However, no functional PTF-1 binding site, which is responsible for pancreas-specific transcription in higher vertebrates, was identified in seabass amylase promoter. Instead a Hepatocyte Nuclear Factor 3 binding site was found to modulate the amylase promoter expression. The evolutionary significance of this divergence in promoter regulation between seabass and mammals requires further studies.

Significance of beta116 His (G18) at alpha1beta1 contact sites for alphabeta assembly and autoxidation of hemoglobin.

The role of heterotetramer interaction sites in assembly and autoxidation of hemoglobin is not clear. The importance of beta(116His) (G-18) and gamma(116Ile) at one of the alpha1beta1 or alpha1gamma1 interaction sites for homo-dimer formation and assembly in vitro of beta and gamma chains, respectively, with alpha chains to form human Hb A and Hb F was assessed using recombinant beta(116His)(-->)(Asp), beta(116His)(-->)(Ile), and beta(112Cys)(-->)(Thr,116His)(-->)(Ile) chains. Even though beta chains (e.g., 116 His) are in monomer/tetramer equilibrium, beta(116Asp) chains showed only monomer formation. In contrast, beta(116Ile) and beta(112Thr,116Ile) chains showed homodimer and homotetramer formation like gamma-globin chains which contain 116 Ile. Assembly rates in vitro of beta(116Ile) or beta(112Thr,116Ile) chains with alpha chains were 340-fold slower, while beta(116Asp) chains promoted assembly compared to normal beta-globin chains. These results indicate that amino acid hydrophobicity at the G-18 position in non-alpha chains plays a key role in homotetramer, dimer, and monomer formation, which in turn plays a critical role in assembly with alpha chains to form Hb A and Hb F. These results also suggest that stable dimer formation of gamma-globin chains must not occur in vivo, since this would inhibit association with alpha chains to form Hb F. The role of beta(116His) (G-18) in heterotetramer-induced stabilization of the bond with oxygen in hemoglobin was also assessed by evaluating autoxidation rates using recombinant Hb tetramers containing these variant globin chains. Autoxidation rates of alpha(2)beta(2)(116Asp) and alpha(2)beta(2)(116Ile) tetramers showed biphasic kinetics with the faster rate due to alpha chain oxidation and the slower to the beta chain variants whose rates were 1.5-fold faster than that of normal beta-globin chains. In addition, NMR spectra of the heme area of these two hemoglobin variant tetramers showed similar resonance peaks, which are different from those of Hb A. Oxygen-binding properties of alpha(2)beta(2)(116His)(-->)(Asp) and alpha(2)beta(2)(116His)(-->)(Ile), however, showed slight alteration compared to Hb A. These results suggest that the beta116 amino acid (G18) plays a critical role in not only stabilizing alpha1beta1 interactions but also in inhibiting hemoglobin oxidation. However, stabilization of the bonds between oxygen and heme may not be dependent on stabilization of alpha1beta1 interactions. Tertiary structural changes may lead to changes in the heme region in beta chains after assembly with alpha chains, which could influence stability of dioxygen binding of beta chains.