The effect of formaldehyde on the oxygen equilibrium of hemoglobin

1. When formaldehyde (0.10 M) is added to solutions of human hemoglobin, the oxygen affinity of the hemoglobin increases considerably (more than tenfold near pH 7). The interaction between hemes of the same hemoglobin molecule decreases, as shown by a drop in the value of n in Hill's equation from 2.9 to 1.5 or less. 2. In the presence of formaldehyde, both n and the oxygen pressure for half-saturation fall gradually as the pH rises in the range from pH 6.2 to 7.2. 3. Some of the effect of formaldehyde on the oxygen equilibrium may be due to combination with sulfhydryl groups of the protein, but nitrogenous groups are probably also involved.     

In vitro effect of nicotine and cotinine on the susceptibility of LDL oxidation and hemoglobin glycosylation

Nicotine, a major component of cigarette smoke, plays an important role in the development of cardiovascular disease and lung cancer in smokers. This study was designed to determine the in vitro effects of nicotine and its metabolite cotinine on the susceptibility of LDL to oxidation and hemoglobin glycosylation. Three different concentrations of each one (10, 15, 25 g/ml) were used. Our data show that nicotine and cotinine are inhibitors for Cu²⁺-induced LDL oxidation but also they increase the glycosylation degree of hemoglobin. Nicotine at final concentrations of (10, 15, 25 g/ml) increases the rate of hemoglobin glycosylation 25, 32 and 47%, respectively, and cotinine at final concentrations increase the rate of glycosylation 8, 10 and 12%, respectively. Therefore promoting hemoglobin glycosylation is one of the alternations caused by smoking that increase risk of cardiovascular disease.     

Reaction of tobacco smoke aldehydes with human hemoglobin

Formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde, and acrolein, all of which are constituents of tobacco smoke, were reacted in 5 mM concentration with the purified major fraction of normal adult human hemoglobin (hemoglobin Ao) in 1 mM concentration. A cigarette smoke condensate, diluted to contain 5 mM total aldehydes, was also reacted with 1 mM hemoglobin Ao. Cationic exchange high-performance liquid chromatography (HPLC) showed that the products formed from simple aliphatic aldehydes, with the exception of formaldehyde, were analogues of those formed from acetaldehyde, earlier shown by us to be imidazolidinone derivatives, that is, cyclic addition products of the N-terminal aminoamide function of α and β chains. Formaldehyde and acrolein produced a heterogeneous mixture of derivatives including crosslinked hemoglobin dimers. The greater proportion of modified hemoglobins produced by condensate aldehydes resembled those formed from acetaldehyde, the most abundant aldehyde in the condensate. A smaller fraction consisted of crosslinked hemoglobin dimers, presumably due to the action of formaldehyde. Mass spectrometric and HPLC analyses of the 2,4-dinitrophenylhydrazones precipitated from the condensate documented the presence of formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, furfsral, and methylfurfural. The toxicity of aldehydes is briefly discussed in the context of the findings of this study.     

A theoretical model to predict the behavior of glycosylated hemoglobin levels

The measurement of glycosylated hemoglobin as a percentage of total hemoglobin is rapidly becoming the standard method of monitoring the average blood sugar level in diabetics for research purposes and may soon become the standard for clinical care and diagnosis. Much speculation exists in the literature about the nature of the glycosylation reaction. Most experimenters expect a linear relationship between the plasma glucose level and percent glycosylated hemoglobin in whole blood; however, a curve of decreasing slope with increasing glucose concentration is found.Here, a reaction model including simple first order kinetics between glucose and hemoglobin and a finite erythrocyte life of 120 days is considered. By carrying out the integration for each erythrocyte cohort followed by an integration combining all cohorts, a curve corresponding to the experimental result is found. In addition, results on expected glycosylated hemoglobin percent as a function of erythrocyte age and plasma glucose concentration are presented as well as a plot of glucose concentration versus glycosylated hemoglobin percent for the 40-day erythrocyte life in mice. All of the results correlate with experimental values in the literature if a rate constant of k = 1·0 × 10^?5dlmg^?1 day is used.The evaluation of a radioactive iron-transferrin experiment in the literature reveals the possibility that the glycosylation reaction begins during erythropoiesis.Finally, a curve is displayed which shows the expected 120-day decay during normoglycemia, of an elevated glycosylated hemoglobin level resulting from a preceding period of constant hyperglycemia.     

PHYSICOCHEMICAL EFFECTS OF ALDEHYDES ON THE HUMAN ERYTHROCYTE

The effects of formaldehyde, acetaldehyde, and glutaraldehyde on human red blood cells were investigated. It was found that (a) The surface negative charge of the erythrocytes at pH 7 was increased 10% by glutaraldehyde, but not by the other two aldehydes. (b) The effect of incomplete fixation of the red blood cells was demonstrated by hemoglobin leakage studies The leakage of hemoglobin subsequent to formaldehyde treatment was especially pronounced Acetaldehyde-fixed cells showed some leakage of hemoglobin after an hour of exposure to the fixative, whereas glutaraldehyde-fixed cells showed no hemoglobin leakage. (c) All three aldehydes caused K⁺ leakage during fixation. The concentrations of K⁺ in the fixing solutions all reached the same level, but whereas the leakage with glutaraldehyde was immediate, that with formaldehyde was more gradual and that with acetaldehyde reached a steady state only after 24 hr. (d) The effects of the aldehydes on red cell deformability and swelling revealed that glutaraldehyde hardened the cells within 15 min, formaldehyde within 5 hr, while acetaldehyde required at least 24 hr to produce appreciable fixation. (e) The hematocrit changes accompanying the fixation process depended upon cell volume changes and loss of deformability.     

Analysis of glycosylation motifs and glycosyltransferases in Bacteria and Archaea

The process of glycosylation has been studied extensively in prokaryotes but many questions still remain unanswered. Glycosyltransferase is the enzyme which mediates glycosylation and has its preference for the target glycosylation sites as well as for the type of glycosylation i.e. N-linked and O-linked glycosylation. In this study we carried out the bioinformatics analysis of one of the key enzymes of pgl locus from Campylobacter jejuni, known as PglB, which is distributed widely in bacteria and AglB from archaea. Relatively little sequence similarity was observed in the archaeal AglB(s) as compared to those of the bacterial PglB(s). In addition we tried to the answer the question of as to why not all the sequins Asp-X-Ser/Thr have an equal opportunity to be glycosylated by looking at the influence of the neighboring amino acids but no significant conserved pattern of the flanking sites could be identified. The software tool was developed to predict the potential glycosylation sites in autotransporter protein, the virulence factors of gram negative bacteria, and our results revealed that the frequency of glycosylation sites was higher in adhesins (a subclass of autotransporters) relative to the other classes of autotransporters.     

Molecular mass of macromolecules and subunits and the quaternary structure of hemoglobin from the microcrustacean Daphnia magna

The molecular masses of macromolecules and subunits of the extracellular hemoglobin from the fresh-water crustacean Daphnia magna were determined by analytical ultracentrifugation, multiangle laser light scattering and electrospray ionization mass spectrometry. The hemoglobins from hypoxia-incubated, hemoglobin-rich and normoxia-incubated, hemoglobin-poor Daphnia magna were analyzed separately. The sedimentation coefficient of the macromolecule was 17.4 +/- 0.1 S, and its molecular mass was 583 kDa (hemoglobin-rich animals) determined by AUC and 590.4 +/- 11.1 kDa (hemoglobin-rich animals) and 597.5 +/- 49 kDa (hemoglobin-poor animals), respectively, determined by multiangle laser light scattering. Measurements of the hemoglobin subunit mass of hemoglobin-rich animals by electrospray ionization mass spectrometry revealed a significant peak at 36.482 +/- 0.0015 kDa, i.e. 37.715 kDa including two heme groups. The hemoglobin subunits are modified by O-linked glycosylation in the pre-A segments of domains 1. No evidence for phosphorylation of hemoglobin subunits was found. The subunit migration behavior during SDS/PAGE was shown to be influenced by the buffer system used (Tris versus phosphate). The subunit mass heterogeneity found using Tris buffering can be explained by glycosylation of hemoglobin subunits. Based on molecular mass information, Daphnia magna hemoglobin is demonstrated to consist of 16 subunits. The quaternary structure of the Daphnia magna hemoglobin macromolecule was assessed by three-dimensional reconstructions via single-particle analysis based on negatively stained electron microscopic specimens. It turned out to be much more complex than hitherto proposed: it displays D4 symmetry with a diameter of approximately 12 nm and a height of about 8 nm.     

Effects of formaldehyde fixation on protein secondary structure: a calorimetric and infrared spectroscopic investigation

We investigated the effects of formaldehyde fixation on the secondary structure of isolated proteins (bovine serum albumin, ribonuclease A, and hemoglobin) using high-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy. Whereas thermograms obtained by scanning calorimetry on unfixed purified proteins demonstrated denaturation transitions in the 70-90 degrees C temperature range, the thermograms showed no denaturation transitions in this temperature range when the proteins had been placed in formaldehyde solutions. Thus, fixation destroyed the denaturation transition of bovine serum albumin, ribonuclease A, and hemoglobin. Infrared spectra obtained on the unfixed and fixed proteins were essentially identical. This demonstrates that the "fixed" proteins retain the secondary structure present before fixation. We therefore conclude that the cross-linking of proteins that occurs in the process of formaldehyde fixation "locks in" the secondary structure of these protein molecules.     

Non-enzymic glycation of proteins: analysis of N-(1-deoxyhexitol-1-yl)amino acids by high-performance liquid chromatography

A method for determining the extent of non-enzymic glycation (originally called "glycosylation") of both lysyl and N-terminal residues of a protein is described. The glycated protein is treated with sodium borohydride, and is then subjected to acid-catalysed hydrolysis. The resulting N-(1-deoxy-D-hexitol-1-yl)amino acids are separated by cation-exchange high-performance liquid chromatography (l.c.), and detected by a post-column reaction with periodate. The method has been applied successfully to samples of human hemoglobin and human serum albumin, for measurement of numbers of valine-attached and of lysine-attached N-(1-deoxy-D-fructos-1-yl) groups per protein molecule.     

The effect of glycosylation on the folding kinetics of erythropoietin

Glycosylation is a common posttranslational modification that generally increases protein solubility and thermodynamic stability. Less is known about how this modification influences protein folding, particularly folding processes involving intermediate species. In the present report, folding comparisons of a nonglycosylated erythropoietin (EPO) mutant are made with the fully glycosylated EPO, which was recently shown to fold by a three-state on-pathway mechanism. The absence of glycosylation did not alter the folding mechanism of EPO but did greatly decrease the stability of the intermediate species, change the rate-limiting step of the folding reaction, and accelerate the folding kinetics to both the intermediate state and the native state. Surprisingly, glycosylation stabilized the intermediate species to a greater extent than it increased the EPO equilibrium stability. These results suggest that glycosylation impedes the latter EPO folding steps rather than accelerating them by biasing particular folding pathways, as previously proposed for folding reactions initiated from unfolded ensembles with minimal residual structure. Due to the specific biological processes modulated by EPO glycosylation, however, there may be little evolutionary pressure to fold on a faster, more direct pathway at the expense of biological function, particularly given the protective role glycosylation has at preventing EPO aggregation. Lastly, evidence that is consistent with glycosylation destabilizing the unfolded state to some degree and contributing to the greater equilibrium stability of the glycosylated EPO is presented.     

N-Glycosylation of the Drosophila neural protein Chaoptin is essential for its stability, cell surface transport and adhesive activity

Glycosylation of proteins can modulate their function in a striking variety of systems, including immune responses, neuronal activities and development. The Drosophila protein, Chaoptin (Chp), is essential for the development and maintenance of photoreceptor cells. This protein is heavily glycosylated, but the possible role of this glycosylation is not well-understood. Here we show that mutations introduced into about 1/3 of 16 potential N-linked glycosylation sites within Chp impaired its cell adhesive activities when expressed in Drosophila S2 cells. Mutation of 2/3 of the glycosylation sites resulted in a marked decrease in Chp protein abundance. These results suggest that N-linked glycosylation of Chp is essential for its stability and activity.     

Mass spectrometry investigation of glycosylation on the NXS/T sites in recombinant glycoproteins

We used a targeted proteomics approach to investigate whether introduction of new N-linked glycosylation sites in a chimeric protein influence the glycosylation of the existing glycosylation sites. To accomplish our goals, we over-expressed and purified a chimeric construct that contained the Fc region of the IgG fused to the exons 7 & 8 of mouse ZP3 (IgG-Fc-ZP3E7 protein). Immunoglobulin heavy chain (IgG-HC protein) was used as control. We then analyzed the IgG-HC and IgG-Fc-ZP3E7 proteins by liquid chromatography-tandem mass spectrometry (LC–MS/MS) and by Western blotting (WB). We concluded that in control experiments, the glycosylation site was occupied as expected. However, in the IgG-Fc-ZP3E7 protein, we concluded that only one out of three NXS/T glycosylation sites is occupied by N-linked oligosaccharides. We also concluded that in the IgG-Fc-ZP3E7 protein, upon introduction of additional potential NXS/T glycosylation sites within its sequence, the original NST/S glycosylation site from the Fc region of the IgG-Fc-ZP3E7 protein is no longer glycosylated. The biomedical significance of our findings is discussed.     

Glycosylation of α-dystroglycan: O-mannosylation influences the subsequent addition of GalNAc by UDP-GalNAc polypeptide N-acetylgalactosaminyltransferases

O-Linked glycosylation is a functionally and structurally diverse type of protein modification present in many tissues and across many species. α-Dystroglycan (α-DG), a protein linked to the extracellular matrix, whose glycosylation status is associated with human muscular dystrophies, displays two predominant types of O-glycosylation, O-linked mannose (O-Man) and O-linked N-acetylgalactosamine (O-GalNAc), in its highly conserved mucin-like domain. The O-Man is installed by an enzyme complex present in the endoplasmic reticulum. O-GalNAc modifications are initiated subsequently in the Golgi apparatus by the UDP-GalNAc polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T) enzymes. How the presence and position of O-Man influences the action of the ppGalNAc-Ts on α-DG and the distribution of the two forms of glycosylation in this domain is not known. Here, we investigated the interplay between O-Man and the addition of O-GalNAc by examining the activity of the ppGalNAc-Ts on peptides and O-Man-containing glycopeptides mimicking those found in native α-DG. These synthetic glycopeptides emulate intermediate structures, not otherwise readily available from natural sources. Through enzymatic and mass spectrometric methods, we demonstrate that the presence and specific location of O-Man can impact either the regional exclusion or the site of O-GalNAc addition on α-DG, elucidating the factors contributing to the glycosylation patterns observed in vivo. These results provide evidence that one form of glycosylation can influence another form of glycosylation in α-DG and suggest that in the absence of proper O-mannosylation, as is associated with certain forms of muscular dystrophy, aberrant O-GalNAc modifications may occur and could play a role in disease presentation.     

Quantitation of lysine-bound glucose of normal and diabetic erythrocyte membranes by HPLC analysis of furosine [ epsilon-N(L-furoylmethyl)-L-lysine]

Non-enzymatic glycosylation of erythrocyte membranes was studied using a non-radioactive and sensitive procedure for specific quantitation of lysine-bound glucose in proteins. About 2 nmol lysine-bound glucose/mg protein were found in ghosts from normal erythrocytes, and this value was about doubled in diabetic patients. In vitro incubation of normal ghosts with glucose gave rise to levels of lysine-bound glucose similar to those found in diabetics. There was a linear correlation between the amount of lysine-bound glucose of total hemoglobin and of membrane proteins. Membrane glycosylation also depended on the age of erythrocytes displaying significantly higher values in old cell populations.     

Hexamethylenetetramine: A powerful and novel inhibitor of gelation of deoxyhemoglobin S

The solubility of deoxyhemoglobin S increases markedly after exposure to hexamethylenetetramine. It has been determined that even at neutral pH hexamethylenetetramine undergoes a slow and slight decomposition into ammonia and formaldehyde. These decomposition products are shown to be responsible for the increase in the solubility of deoxyhemoglobin S. Formaldehyde cross-links the protein and ammonia increases the pH of the buffered hemoglobin solution. The hydrolysis equilibrium between HCHO, NH₃, and hexamethylenetetramine is shifted towards decomposition by the presence of hemoglobin and the reaction is facilitated by reducing agents. Red cells are shown to be readily permeable to hexamethylenetetramine and the potential of this relatively nontoxic chemical for sickle cell disease is discussed.     

Structure, mechanism and engineering of plant natural product glycosyltransferases

Glycosylation is a key mechanism in determining chemical complexity and diversity of plant natural products, and influencing their chemical properties and bioactivities. Uridine diphosphate glycosyltransferases (UGTs) are the central players in these glycosylation processes for decorating natural products with sugars. Crystal structures of plant UGTs have revealed their exquisite architectures and provided the structural basis for understanding their catalytic mechanism and substrate specificity. Structure-based UGT engineering can alter substrate specificity; compromise or enhance catalytic efficiency; and confer reversibility to the glycosylation reaction. This review highlights the structural insights on plant UGTs and successes in glycosylation engineering.     

Mass spectrometry based characterization of Hb Beckman variant in a falsely elevated HbA1c sample

Glycated hemoglobin (HbA_1c) is a ‘gold standard’ biomarker for assessing the glycemic index of an individual. HbA_1c is formed due to nonenzymatic glycosylation at N-terminal valine residue of the β-globin chain. Cation exchange based high performance liquid chromatography (CE–HPLC) is mostly used to quantify HbA_1c in blood sample. A few genetic variants of hemoglobin and post-translationally modified variants of hemoglobin interfere with CE–HPLC-based quantification, resulting in its false positive estimation. Using mass spectrometry, we analyzed a blood sample with abnormally high HbA_1c (52.1%) in the CE–HPLC method. The observed HbA_1c did not corroborate the blood glucose level of the patient. A mass spectrometry based bottom up proteomics approach, intact globin chain mass analysis, and chemical modification of the proteolytic peptides identified the presence of Hb Beckman, a genetic variant of hemoglobin, in the experimental sample. A similar surface area to charge ratio between HbA_1c and Hb Beckman might have resulted in the coelution of the variant with HbA_1c in CE–HPLC. Therefore, in the screening of diabetes mellitus through the estimation of HbA_1c, it is important to look for genetic variants of hemoglobin in samples that show abnormally high glycemic index, and HbA_1c must be estimated using an alternative method.     

Granular Formulations of Steinernema carpocapsae (strain All) (Nematoda: Rhabditida) with Improved Shelf Life

Shelf life (nematode survival) of Steinernema carpocapsae (strain All) nematodes at 21 C in "Pesta" granules, made by a pasta-like process, was increased from 8 to 26 weeks by incorporating low concentrations of formaldehyde. Pesta samples containing an average of 427,000 nematodes/g were prepared with wheat flour (semolina or bread flour), kaolin, bentonite, peat moss, nematode slurry, and formaldehyde (0-1.4% w/w) and were dried to a water content of 23.6-26.9%. Nematodes emerged from Pesta (S. carpocapsae) granules when placed in water or on moist filter paper. Incorporation of 0.2% w/w formaldehyde (nominal; 0.05% by analysis) was optimum for increasing nematode survival in semolina-based Pesta, and also inhibited fungal growth on the granules. Bread flour Pesta samples prepared by formaldehyde addition to the nematode slurry prior to dough preparation, rather than by addition to a mixture of dry ingredients, had longer shelf life. Nematodes recovered from granules made with 0.2% formaldehyde and stored 20 weeks at 21 C caused 100% mortality of wax moth (Galleria mellonella) larvae.     

Glycosylation and functionality of recombinant β-glucocerebrosidase from various production systems

The glycosylation of recombinant β-glucocerebrosidase, and in particular the exposure of mannose residues, has been shown to be a key factor in the success of ERT (enzyme replacement therapy) for the treatment of GD (Gaucher disease). Macrophages, the target cells in GD, internalize β-glucocerebrosidase through MRs (mannose receptors). Three enzymes are commercially available for the treatment of GD by ERT. Taliglucerase alfa, imiglucerase and velaglucerase alfa are each produced in different cell systems and undergo various post-translational or post-production glycosylation modifications to expose their mannose residues. This is the first study in which the glycosylation profiles of the three enzymes are compared, using the same methodology and the effect on functionality and cellular uptake is evaluated. While the major differences in glycosylation profiles reside in the variation of terminal residues and mannose chain length, the enzymatic activity and stability are not affected by these differences. Furthermore, the cellular uptake and in-cell stability in rat and human macrophages are similar. Finally, in vivo studies to evaluate the uptake into target organs also show similar results for all three enzymes. These results indicate that the variations of glycosylation between the three regulatory-approved β-glucocerebrosidase enzymes have no effect on their function or distribution.