New fluorescence of nonenzymatically glucosylated human serum albumin

Glucosylated human serum albumin (G-HSA) obtained under incubation with glucose at 37 degrees C for 8 days showed a new fluorescence with a maximum at 430 nm, resulting in quenching of the fluorescence of only one tryptophan residue on HSA. The quantum yield of new fluorescence is 0.024 at 25 degrees C. The analysis of the excitation spectra allowed us to conclude the absence of energy transfer. In G-HSA, non-disulfide cross-linking hexamer was confirmed by SDS-PAGE.     

Antibodies to nonenzymatically glucosylated albumin in the human serum

The existence of antibodies to nonenzymatically glucosylated albumin was investigated in nondiabetic and diabetic subjects. The sera from both the nondiabetic and the diabetic subjects were shown to contain the proteins which bound to reductively glucosylated albumin. An enzyme-linked immunosorbent assay demonstrated that the antibodies specific for reductively glucosylated albumin existed in the sera containing the binding proteins. For binding the antibodies glucitollysine as the glucose adduct in reductively glucosylated albumin was an effective competitor. The hexose alcohol epimers glucitol and mannitol were also effective competitors compatible with glucitollysine. Our results suggest that the antibodies to reductively glucosylated albumin are widely present not only in the diabetic subjects but also in the nondiabetic subjects and cross-react with the hexose alcohol.     

Spontaneous degradation of hemoglobin and egg albumin

Hemolysates of red cells of the horse, after removal of stroma and prolonged dialysis, released free amino acids on standing. A similar release of amino acids was not found when a solution of purified hemoglobin A was allowed to age, but a small number of peptides were found. Crude and purified egg albumin, when allowed to stand in solution, released free amino acids in a manner similar to that of the hemolysate of red cells. Quantitative data on the release of free amino acids are reported in this communication. The variety and distribution of the amino acids make it unlikely that this hydrolytic process is solely the result of contamination by proteolytic enzymes. The proposal is made that this property results from an intrinsic enzyme-like activity associated with the sulfhydryl groups of these proteins.     

Glycated albumin and glycated hemoglobin levels in normal pregnancy

Glycated albumin levels showed a progressive increase during normal pregnancy. The mean values (mole hexose/mole protein) were 1.68 +/- 0.27 (n = 15) in nonpregnant women, 1.83 +/- 0.21 (n = 11) in first trimester, 2.00 +/- 0.41 (n = 13) in second trimester, and 2.42 +/- 0.49 (n = 15) in third trimester. Glycated hemoglobin levels indicated a biphase pattern with low values at midpregnancy (controls 0.29 +/- 0.05, first trimester 0.30 +/- 0.04, second trimester 0.27 +/- 0.05, and third trimester 0.33 +/- 0.04). The data suggest that glycated albumin reflects the decreased glucose tolerance in pregnancy better that glycated hemoglobin levels. The reasons for the differing pattern of the two glycated proteins are discussed.     

Crystal structure of an inactive duck delta II crystallin mutant with bound argininosuccinate

Delta-crystallin, the major soluble protein component of avian and reptilian eye lenses, is highly homologous to the urea cycle enzyme, argininosuccinate lyase (ASL). In duck lenses, there are two highly homologous delta crystallins, delta I and delta II, that are 94% identical in amino acid sequence. While delta II crystallin has been shown to exhibit ASL activity in vitro, delta I is enzymatically inactive. The X-ray structure of a His to Asn mutant of duck delta II crystallin (H162N) with bound argininosuccinate has been determined to 2.3 A resolution using the molecular replacement technique. The overall fold of the protein is similar to other members of the superfamily to which this protein belongs, with the active site located in a cleft formed by three different monomers in the tetramer. The active site of the H162N mutant structure reveals that the side chain of Glu 296 has a different orientation relative to the homologous residue in the H91N mutant structure [Abu-Abed et al. (1997) Biochemistry 36, 14012-14022]. This shift results in the loss of the hydrogen bond between His 162 and Glu 296 seen in the H91N and turkey delta I crystallin structures; this H-bond is believed to be crucial for the catalytic mechanism of ASL/delta II crystallin. Argininosuccinate was found to be bound to residues in each of the three monomers that form the active site. The fumarate moiety is oriented toward active site residues His 162 and Glu 296 and other residues that are part of two of the three highly conserved regions of amino acid sequence in the superfamily, while the arginine moiety of the substrate is oriented toward residues which belong to either domain 1 or domain 2. The analysis of the structure reveals that significant conformational changes occur on substrate binding. The comparison of this structure with the inactive turkey delta I crystallin reveals that the conformation of domain 1 is crucial for substrate affinity and that the delta I protein is almost certainly inactive because it can no longer bind the substrate.     

Polynitroxyl hemoglobin: a pharmacokinetic study of covalently bound nitroxides to hemoglobin platforms

Adding antioxidant activities to hemoglobin-based oxygen carriers (HBOCs) represents a means of reducing cell-free hemoglobin-mediated oxidative cascades. We have covalently bound nitroxides, a class of antioxidant enzyme mimetics, to HBOCs. The objectives of this study were (1) to evaluate the pharmacokinetic (PK) effects of administering nitroxide covalently bound to HBOCs compared to those of free nitroxide coadministered with HBOCs and (2) to elucidate the effects of differing molecular weight HBOCs on the PK of bound nitroxide in a conscious guinea pig model of 25% blood exchange transfusion. Two HBOC platforms were used, intramolecular cross-linked hemoglobin (XLHb) and dextran polymerized/conjugated XLHb (PolyHb). Polynitroxylation was achieved by reacting 4-(2-bromoacetamido)-2,2,6,6,-tetramethylpiperidine-1-oxyl with XLHb or PolyHb to form polynitroxylated XLHb and polynitroxylated PolyHb, respectively, whereas a physical mixture of XLHb or PolyHb with 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl was prepared to reflect a molar equivalence to HBOC-bound nitroxide. Plasma concentrations of two redox states, nitroxide and hydroxylamine, were determined by electron paramagnetic resonance spectroscopy. Results are presented to illustrate the influence of covalent labeling and HBOC molecular weight on nitroxide PK. The therapeutic potential of polynitroxylation of HBOCs as it relates to observations from the current and previously reported studies is discussed.     

Expression of monocyte chemoattractant protein-1 by nonenzymatically glycated albumin (Amadori adducts) in vascular smooth muscle cells

The biological effects of Amadori adducts that are early nonenzymatically glycated protein on vascular cells were poorly defined. We examined the effect of glycated serum albumin (GA) on the expression of monocyte chemoattractant protein-1(MCP-1) that is an important chemokine recruiting monocyte to blood vessel. GA increased MCP-1 mRNA expression with a peak after 3 h of stimulation. The induction of MCP-1 by GA was dose-dependent. The MCP-1 mRNA expression by GA was completely inhibited by PD98059 and genistein that inhibit mitogen activated protein (MAP) kinase kinase and tyrosine kinase, respectively. N-Acetylcysteine, a potent antioxidant, also suppressed the GA-induced MCP-1 expression. These results suggest that GA induces production of reactive oxygen species and activates tyrosine kinase and MAP kinase in VSMC. Activation of these signals results in MCP-1 expression. GA-induced MCP-1 expression may be one of the mechanisms by which the diabetic patients suffer from accelerated atherosclerosis.     

Characterization of methylene diphenyl diisocyanate-haptenated human serum albumin and hemoglobin

Protein haptenation by polyurethane industrial intermediate 4,4′-methylene diphenyl diisocyanate (MDI) is thought to be an important step in the development of diisocyanate (dNCO)-specific allergic sensitization; however, MDI-haptenated albumins used to screen specific antibody are often poorly characterized. Recently, the need to develop standardized immunoassays using a consistent, well-characterized dNCO-haptenated protein to screen for the presence of MDI-specific IgE and IgG from workers’ sera has been emphasized and recognized. This has been challenging to achieve due to the bivalent electrophilic nature of dNCOs, leading to the capability to produce multiple cross-linked protein species and polymeric additions to proteins. In the current study, MDI was reacted with human serum albumin (HSA) and hemoglobin (Hb) at molar ratios ranging from 1:1 to 40:1 MDI/protein. Adducts were characterized by (i) loss of available 2,4,6-trinitrobenzene sulfonic acid (TNBS) binding to primary amines, (ii) electrophoretic migration in polyacrylamide gels, (iii) quantification of methylene diphenyl diamine following acid hydrolysis, and (iv) immunoassay. Concentration-dependent changes in all of the above noted parameters were observed, demonstrating increases in both number and complexity of conjugates formed with increasing MDI concentrations. In conclusion, a series of bioanalytical assays should be performed to standardize MDI–antigen preparations across lots and laboratories for measurement of specific antibody in exposed workers that in total indicate degree of intra- and intermolecular cross-linking, number of dNCOs bound, number of different specific binding sites on the protein, and degree of immunoreactivity.     

EPR study of non-covalent spin labeled serum albumin and hemoglobin

Electron Paramagnetic Resonance (EPR) was used to investigate the Tempyo spin label (3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy) as a report group for the interactions and the conformational changes of lyophilized bovine serum albumin (BSA) and bovine hemoglobin (BH), as function of pH values in the range 2.5-11. The EPR spectra are similar with those of other non-covalently spin label porphyrins in frozen solution at very low temperatures. This behavior indicated a possible spin-spin interaction between the hemic iron and the nitroxide group. The changes in the EPR spectra as function of the pH are discussed in terms of conformational changes of the proteins. Spectral simulations and magnetic EPR parameters reveal the following: (i) one single paramagnetic species, with Gaussian line shape, was used for the best fits of experimental spectra in the case of serum albumin samples; and (ii) a weighted sum of Lorentzian and Gaussian line shape in the case of hemoglobin samples. The representation of correlation time vs. pH, reveals a dependence of degree of immobilization of spin label on the conformational changes of proteins in acidic and basic environment.     

Steady-state fluorescence emission from the fluorescent probe, 5-iodoacetamidofluorescein, bound to hemoglobin

In the past, fluorescence emission from an extrinsic fluorophore bound to heme-proteins would only be studied with the removal of the heme since fluorescence from the fluorophore could not be detected using right-angle optics. Using front-face fluorometry, a significant steady state emission signal originating from the probe bound to hemoglobin is detected. This is the first report of the detection of extrinsic fluorescence of a probe bound to a heme-protein. We also demonstrate that the extrinsic probe, 5-iodoacetamidofluorescein, is covalently bound to hemoglobin, specifically at beta 93 Cysteine. Ligand binding results in a change in the fluorophore fluorescence intensity as predicted by hemoglobin crystallographic studies. Efficiency of energy transfer measurements are made.     

Glycated hemoglobin and albumin reflect nestling growth and condition in American kestrels

Blood chemistry can be used to assess physiological state and condition. Levels of glycated hemoglobin (GHb), which integrates blood glucose levels over a period of weeks, may provide a way to assess resource intake. I tested whether GHb reflects offspring quality by comparing growth rates of nestling American kestrels (Falco sparverius) with GHb levels at 24 days of age. Nestlings that gained structural size faster had higher levels of GHb than did slower growing nestlings. There was no difference in GHb levels between males and females, although females are larger. In addition, I tested whether albumin levels, a measure of protein storage, were correlated with nestling growth and body condition (reflected in residual body mass). Larger individuals, measured by both absolute body mass and by residual body mass, had larger levels of albumin. This was due in part to females having higher albumin levels. Interestingly, there was no correlation between GHb and albumin, suggesting that both measures are necessary to assess physical condition in nestling kestrels. These results suggest that blood chemistry can reflect offspring condition measures and may provide a way to assess offspring quality that reflects conditions experienced by offspring over longer periods through measurements at a single time point.     

Antioxidant activity of liver growth factor,a bilirubin covalently bound to albumin

We previously reported that treatment of spontaneously hypertensive rats (SHR) with liver growth factor (LGF), an albumin–bilirubin complex with a covalent bond, reduces blood pressure, improves nitric oxide (NO)-dependent vasodilatation, and exerts vascular antifibrotic actions. Because bilirubin, albumin, and albumin-bound bilirubins have antioxidant properties, we hypothesize that LGF might exert its cardiovascular actions through an antioxidant mechanism. We have tested in vitro the capacity of LGF to scavenge ABTS cation and peroxyl and hydroxyl radicals and to protect vascular NO from degradation by superoxide anion. We have also compared the antioxidant capacity of LGF with that of its molecular components albumin and bilirubin and the reference antioxidant trolox. LGF exhibited antioxidant capacity against all free radicals tested at lower concentrations than albumin, bilirubin, and trolox. LGF, bilirubin, and albumin were also able to protect endothelial NO from superoxide anion degradation in a fashion similar to that of superoxide dismutase or tiron, but at much lower concentrations. These data, together with our previous results in SHR, suggest that LGF might exert its cardiovascular regenerative actions, at least in part, through an antioxidant mechanism and that LGF could be a relevant circulating antioxidant in situations of oxidative stress.     

Comparison of PEG-modified albumin and hemoglobin in extreme hemodilution in the rat.

We have reported a new polyethylene glycol (PEG)-modified, hemoglobin-based O2 carrier (MP4) with novel properties, including a large molecular excluded volume and low PO2 necessary to obtain 50% O2 (approximately 6 Torr). To evaluate the ability of MP4 to transport O2, we compared it with PEG-modified albumin (MPA) using the identical chemistry of attachment of PEG chains. The resulting solutions were well matched with respect to all physical properties except that MP4 is an O2 carrier, whereas MPA is not. An additional solution, 10% pentastarch, was matched with the PEG-modified proteins with regard to oncotic activity and viscosity but does not contain PEG. The model used to evaluate O2 transport was continuous exchange transfusion in the rat until the hematocrit was virtually unmeasurable. Objective end points included survival and the onset of anaerobic metabolism, signaled by acid-base derangement and accumulation of lactic acid. Continuous exchange transfusion of 2.5 blood volumes in rats (n=5 in each treatment group) was carried out over 60 min, such that the final hematocrit was between 0 and 5% in all animals. Animals were observed for an additional 70 min, when survivors were killed. Overall survival for the MP4 animals was 100%; no animal that received either pentastarch or MPA survived. The hematocrit at which lactic acid began to rise was approximately 14.8% in both pentastarch and MPA animals and 7.4% in the animals that received MP4. In all groups, the total hemoglobin was approximately 5 g/dl at this point. We conclude that, despite its low PO2 necessary to obtain 50% O2, MP4 effectively substitutes for red blood cell hemoglobin in its ability to oxygenate tissues in extreme hemodilution.     

Existence of a bioactive lipid, cyclic phosphatidic acid, bound to human serum albumin

A novel bioactive lipid, cyclic phosphatidic acid (cPA), was identified in lipids bound to human serum albumin. A cPA fraction was extracted and purified from human serum albumin by use of a combination of preparative TLC and HPLC. Electrospray ionization mass spectrometry of the purified fraction showed molecular ions corresponding to cPA, which was composed of some different fatty acid species. The most abundant component was identified as palmitoyl-cPA by tandem mass spectrometry using collision-induced dissociation. These data have established that cPA is a naturally occurring lipid bound to human serum albumin.     

Fatty acids bound to alpha-fetoprotein and albumin during rat development

The time-course levels and composition of the fatty acids bound to rat alpha-fetoprotein (AFP) and albumin from several sources, were determined throughout development, and related to the intake of lipids from milk and the compositional changes in brain and liver fatty acids. The major fatty acids bound to AFP were acids bound to AFP were polyunsaturated and mainly docosahexaenoic acid (22:6(n-3], either from fetal serum (23.1%) or whole fetuses (21.6%), whereas palmitic (34.1%) and oleic (29.9%) acids were the main acids bound to albumin from the same sources. Amniotic fluid AFP contained less fatty acids (0.8 mol/mol protein) than that of fetal serum (1.4 mol/mol protein), and especially noticeable was a reduced amount of 22:6 (9.6%). Both AFP-concanavalin A microforms showed identical fatty acid composition. Levels of 22:6 bound to AFP decreased quickly after birth until a minimum at 8-10 days, increasing moderately thereafter. This minimum is coincident in time with a maximal accumulation of this fatty acid by brain and a loss of 22:6 by liver. Except for colostrum, levels of 22:6 in milk lipids were low and fairly constant, but always greater than those of its precursor, linolenic acid (18:3 (n-3]. These results support a specialized role of AFP in the plasma transport and tissue delivery of polyunsaturated fatty acids, and mainly docosahexaenoic acid.     

Effect of binding to hemoglobin and albumin on pyridoxal transport and metabolism

Scatchard plot analysis indicated that pyridoxal binds to hemoglobin more than twice as tightly as it does to serum albumin. Comparison of the formation constants for hemoglobin and albumin, using standard competitive binding equations, indicated that the distribution ratio for pyridoxal between erythrocytes and plasma should be 6.5:1. This distribution was approximately the same as that observed when pyridoxal was incubated with whole human blood, suggesting that these two proteins are the primary determinants of the pyridoxal distribution in whole blood. With in situ perfused rat liver the uptake of [3H] pyridoxal from the perfusate was reduced by the inclusion of erythrocytes in the perfusate. This was reflected in the decreased production of 4-pyridoxic acid by the perfused liver from 3.8% to 1.2% of the dose by the addition of erythrocytes to the perfusate. The major labeled metabolites found in the liver were pyridoxal phosphate, pyridoxamine phosphate, and 4-pyridoxic acid for both types of perfusion. In intact animals, reduction of the erythrocytes concentrations to hematocrits of 30-40% increased the recovery in the urine of 3H from administered [3H] pyridoxal from control values of 27-35% to 40-50% of the dose within 48 h. Half of the label in urinary metabolites was in 4-pyridoxic acid.