The effect of non-enzymatic glycation on the unfolding of human serum albumin

We monitored the unfolding of human serum albumin (HSA) and glycated human serum albumin (gHSA) subjected to guanidine hydrochloride (GndHCl) by using fluorescence and circular dichroism (CD) spectroscopy. A two-state model with sloping baselines best described the Trp-214 fluorescence unfolding measurements, while a three-state model best described the far-UV CD unfolding data. Glycation of HSA increased the [D](50%) point by approximately 0.20M. This corresponded to an increase in the free energy of unfolding of gHSA relative to HSA of 2.6kJ/mol. The intrinsic fluorescence of Trp-214 in gHSA is 0.72 of that of HSA and the far-UV CD spectrum of gHSA is nearly identical to that of HSA. These results showed that glycation altered the local structure around Trp-214 while not significantly impacting the secondary structure, and this alteration translated into an overall change in the stability of gHSA compared to HSA.     

Conformational analysis of human serum albumin and its non-enzymatic glycation products using monoclonal antibodies

Monoclonal antibodies (mAbs) were prepared to analyse the conformation of human serum albumin (HSA) and its non-enzymatic glycation (NEG) products. We first determined the epitopes of the mAbs using HSA subdomains expressed on the surface of yeast. Each mAb was classified as belonging to one of two groups; Type I mAbs which recognized a single subdomain structure and Type II mAbs which bound to plural subdomains. We analysed the pH-dependent conformational change in HSA. We found that one Type II mAb, HAy2, detected the normal to base form (N-B) transition while the other did not, suggesting that N-B transition occurred around Domain I accompanied by topological isomerization of subdomains without changing the subdomain structure itself. Next, we analysed the conformations of the NEG products. Since all mAbs reacted with the early NEG products, no structural change was thought to have occurred in the early NEG products. On the other hand, only a Type I mAb, HAy1, had full binding activity with the advanced glycation end products (AGE) while the other mAbs had lost or had diminished activity, suggesting that the over-all tertiary structure of HSA was altered except for a subdomain, sDOM Ia, in AGE.     

Papaverine increases human serum albumin glycation

Glycation is a non-enzymatic reaction that is initiated by the primary addition of sugars to amino groups of proteins. In the early phase of glycation, the synthesis of intermediates leads to formation of Amadori compounds. In the last phase, advanced glycation end products (AGE) are irreversibly formed following a complex cascade of reactions. It has recently been shown that glycation also affects diabetes-related complications and Alzheimer’s disease. In this study, human serum albumin at a concentration of 10 mg/ml was incubated in PBS with 40 mM of glucose and in different concentrations of papaverine (25, 100, 250, 500 μM) for 42 days at 37 °C. HSA with no additives as well as with glucose 40 mM were incubated as a control and as a glycated sample, respectively. Following the incubation, the samples were prepared for circular dichroism, fluorescence and absorbance techniques. The results showed that in presence of papaverine and glucose, the glycation of HSA increased notably compared with the glycated sample. In conclusion, in this work, we showed that papaverine affects HSA and increases its glycation level.     

In vitro glycation of human serum albumin by dihydroxyacetone and dihydroxyacetone phosphate

Amino groups in proteins can non-enzymatically react with reducing sugars to generate a structurally diverse group of compounds referred to as advanced glycation end products (AGEs). The in vivo formation of AGEs contributes to some of the complications of diabetes including atherosclerosis, cataract formation, and renal failure. The formation of AGEs is dependent on both sugar and protein concentrations. Increases in temperature, pH, and exposure time of sugars to the proteins also play a significant role in the rate of AGE formation. This study focuses on the use of a combination of analytical techniques to study the in vitro AGE formation of HSA with dihydroxyacetone phosphate (DHAP), a ketose generated during glycolysis, and its dephosphorylated analog, dihydroxy acetone (DHA), commonly used as a browning reagent in skin tanning preparations. The extent of AGE formation was affected by DHAP and DHA concentrations and by the duration of HSA exposure to these glycating agents. Increases in temperature and pH sped the glycation process and enhanced the formation of the AGEs of HSA. MALDI-TOF mass spectroscopic data provided a reliable result to evaluate the extent of the AGE formation.     

The in vitro glycation of human serum albumin in the presence of Zn(II)

Amino groups of human serum albumin (HSA) can react non-enzymatically with carbonyl groups of reducing sugars to form advanced glycation end products (AGEs). These AGEs contribute to many of the chronic complications of diabetes including atherosclerosis, cataract formation and renal failure. The current study focused on in vitro non-enzymatic reactivity of glyceraldehyde (GA) and methylglyoxal (MG) with HSA and evaluated the rate and extent of AGE formation in the presence of varied concentrations of Zn(II). At normal physiological conditions, GA and MG readily react with HSA. The presence of Zn(II) in HSA-GA or HSA-MG incubation mixtures reduced AGE formation. This finding was confirmed by UV and fluorescence spectrometry, HPLC techniques, and matrix assisted laser desorption ionization mass spectrometry (MALDI-TOF). HPLC studies revealed decreased adduct formation of the glycated protein in the presence of Zn(II). The inhibition of AGE formation was intense at elevated Zn(II) concentrations. The results of this study suggest that Zn(II) may prove to be a potent agent in reducing AGE formation.     

A study on human serum albumin influence on glycation of fibrinogen

Although in vivo glycation proceeds in complex mixture of proteins, previous studies did not take in consideration the influence of protein–protein interaction on Maillard reaction. The aim of our study was to test the influence of human serum albumin (HSA) on glycation of fibrinogen. The isotopic labeling using [¹³C₆] glucose combined with LC-MS were applied as tool for identification possible glycation sites in fibrinogen and for evaluation the effect of HSA on the glycation level of selected amino acids in fibrinogen.     

L-Arginine inhibits in vitro nonenzymatic glycation and advanced glycosylated end product formation of human serum albumin

Summary L-Arginine (Arg) has a structure similar to that of aminoguanidine (AG) and may inhibit glycation and advanced glycosylated end product (AGE) formation. Human serum albumin (HSA) (100mg/ml) was incubated for 2 weeks with glucose (200mM) at 37°C or with glucose and equimolar concentrations of Arg, N--acetyl Arg, or AG with or without 25mM diethylenetriaminepentaacetic acid (DTPA). In the absence of DTPA, electrospray ionization mass spectrometry showed a 70% reduction of covalently bound glucose in the presence of Arg and a 30% reduction with AG. Digestibility by trypsin of HSA incubated with glucose and Arg was similar to that of HSA incubated alone. This suggests less covalent modification of HSA in the presence of Arg as compared with the absence of Arg. When incubations contained DTPA, autoradiography showed less¹⁴C labeling of HSA subunits in the presence of Arg and AG. When the-amino group of Arg was blocked with an acetyl group, labeling was similar to that of HSA incubated with glucose, suggesting involvement of the-amino group in the inhibition. Fluorescence of HSA at ex₃₇₀ and em₄₄₀ was reduced with Arg, but AG was more effective than Arg. These results suggest that Arg, like AG, can inhibit glycation and AGE formation.Presented in part at the FASEB meeting, Atlanta, GA, 1991.     

Detergency effects of nanofibrillar amyloid formation on glycation of human serum albumin

The prolonged glycation of human serum albumin (HSA) results in significant changes in its structure. The identity of these structural changes and the influence of carbohydrates on these changes require further study. Here, we evaluated structural changes and amyloid formation of HSA upon incubation with Glc, Fru, or Rib. Fluorescence spectrophotometry, surface tension analysis, and transmission electron microscopy (TEM) were utilized to evaluate the structures of glycated HSA. The physicochemical properties including excess free energy, protein adsorption at the air-water interface, critical aggregation concentration (CAC), and surface activity indicated an increase in hydrophobicity and partial unfolding of HSA structure upon glycation. Thus, it appears that AGE products can act as detergents. Incubation of HSA with these sugars after 20 wks induced significant amyloid nanofibril formation. Together these results indicate that prolonged glycation of HSA is associated with a transition from helical structure to beta-sheet (amyloid formation).     

Hyperglycemia and non-enzymatic glycation of serum and tissue proteins in chickens

The objectives of these studies were to determine whether elevated plasma glucose concentrations in broiler breeder chickens (200–250 mg/dl) can result in the non-enzymatic attachment of glucose to serum proteins (fructosamine) and eventual cross-linking of tissue proteins (basement membrane thickness), and to investigate the effects of a factor that may influence this cross-linking process. In response to feeding the satiety factor calcium propionate (CaP, 1.7%), plasma glucose and fructosamine concentrations were increased (P < 0.05) from 1 to 9 weeks of age, whereas concentrations of plasma glucose and fructosamine in feed-restricted chicks were reduced for the first 7 weeks after hatch. In a second study, the age-related increase in kidney capillary basement membrane thickness was prevented (P < 0.05) by feeding the cross-linking inhibitor aminoguanidine (AG, 800 ppm) to 30-week-old broiler breeder hens for 34 weeks.The results from these studies suggest that concentrations of plasma glucose in chickens may, in fact, be exerting long-term detrimental effects on tissue proteins, which can be ameliorated by factors that limit the cross-linking reaction.     

The subsequent effect of interaction between Co(2+) and human serum albumin or bovine serum albumin.

A notable hysteretic effect has been observed in the interaction of Co(II) with human serum albumin (HSA) or bovine serum albumin (BSA) using UV-Visible spectrometry at physiological pH (7.43), which shows that the binding between Co(II) and HSA or BSA may induce a slow transition of HSA or BSA from the conformation of weaker affinity for Co(II) to one of stronger affinity (A-B transition). The rate constants and activation parameters of this transition were measured and are discussed. It is inferred that such a conformation transition may occur due to the binding of the first Co(II) ion with the peptide segment of N-terminal residues 1-3, which results in a 'hinged movement' of the relatively hydrophobic 'valley' in the IA subdomain. This process leads to a slow conformational transition in the albumins, makes the other binding sites of Co(II) exposed, and shows a positive cooperativity effect. The LMCT (ligand-to-metal charge transition) bands of the Co(II)-HSA and Co(II)-BSA systems also show a kind of hypochromic effect featuring a dipole-dipole interaction mechanism. This phenomenon is rarely reported.     

Kinetics of the glycation of bovine serum albumin by mannose and fucose in vitro

Glycation of bovine serum albumin was measured for mannose and fucose at 37 degrees C. Mannose as well as fucose demonstrated an initial rapid increase in rate of formation of total adducts followed by a slower secondary reaction. The equilibrium constant for Schiff base formation was almost two times larger for mannose than fucose, although the Schiff base formed by fucose rearranged 1.5 times faster than that for mannose. Both sugars showed parallel lines for the formation of total and acid stable products after three hours. Discussion integrates new mechanistic data with previously suggested mechanisms.     

The effect of glycation on the structure, function and biological fate of human serum albumin as revealed by recombinant mutants

Recombinant wild-type human serum albumin (rHSA), the single-residue mutants K199A, K439A and K525A and the triple-residue mutant K199A/K439A/K525A were produced using a yeast expression system. Portions of the rHSA were glycated to different degrees (2.5-250 mM D-glucose). As detected by far-UV and near-UV CD, intrinsic tryptophan-fluorescence and probed by 1,1'-bis(4-anilino)naphthalene-5,5-disulfonic acid, the single-residue mutations had no effect on albumin conformation, whereas the triple-residue mutation and glycation caused conformational changes. The triple-residue mutation and glycation had comparable increased effects on high-affinity binding of warfarin (site I), but decreased effects on high-affinity binding of dansylsarcosine (site II) and the esterase-like activity of albumin. The relation between plasma half-lives in rats were found to be glycated rHSA (50 mM glucose)相似文献   

Suppression of agglomeration of ciprofloxacin-loaded human serum albumin nanoparticles

The present study is aimed at developing and exploring the use of pectin in suppression of agglomeration of ciprofloxacinloaded human serum albumin (HSA) nanoparticles. The HSA-pectin nanoparticles loaded with ciprofloxacin were prepared by the pH-coacervation method, and various physicochemical parameters such as particle size, morphology, ζ-potential, electrolyte-induced flocculation, pH-dependent ζ-potential, drug loading, in vitro drug release, and stability of nanoparticles, were evaluated. The size of the HSA-pectin nanoparticles (F3) was found to be 180 to 290 nm. The HSA nanoparticles were modified with pectin when the critical flocculation concentration of nanoparticles in Na₂SO₄ solution was increased from 0.3 M to 0.9 M. The isoelectric points of the formed nanoparticles were found to be relatively lower between pH values 3 and 6. Pectin may be used as a pharmaceutical additive for the suppression of particle agglomeration in HSA nanoparticles, and the effect may be attributed to the pectin segments present on the surface of nanoparticles. Published: March 2, 2007     

Spectroscopic studies of the effects of glycation of human serum albumin on L-Trp binding

Modification of proteins by nonenzymatic glycation is one of the underlying factors that contribute to the development of the complications of diabetes. Human serum albumin (HSA) is one of the major targets of interaction with glucose through the Maillard reaction. The effects of 1 and 5 mg/ml glucose concentrations, which are consistent with blood glucose levels found in diabetic patients, on human serum albumin were studied by circular dichroism and fluorescence spectroscopy in sodium phosphate buffer, pH 7.4. Partial denaturation and changes in the structural integrity of HSA are caused by glycation at lower (1 mg/ml) and higher (5 mg/ml) concentrations of glucose. To study the relationship between structure and function, we investigated the interaction of L-tryptophan (L-Trp) with glycated and non-glycated HSA. The results showed that L-Trp, as the only free amino acid that substantially binds to HSA, has a lower affinity for the glycated form (especially at low concentrations of glucose) than for non-glycated HSA.     

Effect of (-)-epigallocatechin-3-gallate on glucose-induced human serum albumin glycation

Abstract

(-)-Epigallocatechin-3-gallate (EGCg) is a naturally occurring polyphenol found in plant-based foods and beverages such as green tea. Although EGCg can eliminate carbonyl species produced by glucose autoxidation and thus can inhibit protein glycation, it is also reported to be a pro-oxidant that stimulates protein glycation in vitro. To better understand the balance between antioxidant and pro-oxidant features of EGCg, we evaluated EGCg-mediated bioactivities in a human serum albumin (HSA)/glucose model by varying three different parameters (glucose level, EGCg concentration, and time of exposure to EGCg). Measurements of glycation-induced fluorescence, protein carbonyls, and electrophoretic mobility showed that the level of HSA glycation was positively related to the glucose level over the range 10–100 mM during a 21-day incubation at 37°C and pH: 7.4. Under mild glycemic pressure (10 mM), long exposure to EGCg enhanced HSA glycation, while brief exposure to low concentrations of EGCg did not. Under high glycemic pressure (100 mM glucose), long exposure to EGCg inhibited glycation. For the first time we showed that brief exposure to EGCg reversed glycation-induced fluorescence, indicating a restorative effect. In conclusion, our research identified glucose level, EGCg concentration, and time of exposure as critical factors dictating EGCg bioactivities in HSA glycation. EGCg did not affect HSA glycation under normal physiological conditions but had a potential therapeutic effect on HSA severely damaged by glycation.     

Comparison of bovine serum albumin glycation by ribose and fructose in vitro and in vivo

Advanced glycation end products (AGEs) play a critical pathogenic role in the development of diabetic complications. Recent studies have shown that diabetes is associated with not only abnormal glucose metabolism but also abnormal ribose and fructose metabolism, although glucose is present at the highest concentration in humans. The glycation ability and contribution of ribose and fructose to diabetic complications remain unclear. Here, the glycation ability of ribose, fructose and glucose under a mimic physiological condition, in which the concentration of ribose or fructose was one-fiftieth that of glucose, was compared. Bovine serum albumin (BSA) was used as the working protein in our experiments. Ribose generated more AGEs and was markedly more cytotoxic to SH-SY5Y cells than fructose. The first-order rate constant of ribose glycation was found to be significantly greater than that of fructose glycation. LC-MS/MS analysis revealed 41 ribose-glycated Lys residues and 12 fructose-glycated residues. Except for the shared Lys residues, ribose reacted selectively with 17 Lys, while no selective Lys was found in fructose-glycated BSA. Protein conformational changes suggested that ribose glycation may induce BSA into amyloid-like monomers compared with fructose glycation. The levels of serum ribose were correlated positively with glycated serum protein (GSP) and diabetic duration in type 2 diabetes mellitus (T2DM), respectively. These results indicate that ribose has a greater glycation ability than fructose, while ribose largely contributes to the production of AGEs and provides a new insight to understand in the occurrence and development of diabetes complications.     

Forskolin-loaded human serum albumin nanoparticles and its biological importance

Abstract

In this study, forskolin-loaded human serum albumin nanoparticles (FR-HSANPs) were successfully prepared by incorporation and affinity-binding methods. FR-HSANPs were characterized by transmission electron microscope that most of them are circular in shape and size is around 340?nm. The drug loading was more than 88% and further sustained release profiles were observed as it is 77.5% in 24?h time. Additionally, the cytotoxicity results with HepG2 cells indicated that FR-HSANPs showed significantly higher cytotoxicity and lower cell viability as compared to free forskolin (FR). Furthermore, to understand the binding mechanism of human serum albumin (HSA) with forskolin resulted from fluorescence quenching as a static mechanism and the binding constant is 6.26?±?0.1?×?10⁴ M^?1, indicating a strong binding affinity. Further, association and dissociation kinetics of forskolin–HSA was calculated from surface plasmon resonance spectroscopy and the binding constant found to be K_forskolin = 3.4?±?0.24?×?10⁴ M^?1 and also fast dissociation was observed. Further, we used circular dichroism and molecular dynamics simulations to elucidate the possible structural changes including local conformational changes and rigidity of the residues of both HSA and HSA–forskolin complexes.

Communicated by Ramaswamy H. Sarma