Structural changes in bovine lens crystallins induced by ascorbate, metal, and oxygen

Ascorbate, Fe3+, or Cu2+ and oxygen induced the oxidation of bovine lens crystallins. The modifications mimicked those that occur in the lens with aging. The modifications included the formation of nondisulfide crosslinks in alpha- and beta H-crystallin and the cleavage of alpha-, beta H-, and the low molecular weight crystallin fractions. In all three fractions, there was a loss of the more basic protein species and an increase in the more acidic species. Nontryptophan fluorescence with emission spectra between 400 and 500 nm was produced in beta H-crystallin. Cu2+ was less effective than Fe3+ in catalyzing the modification of beta H- and gamma-crystallin. Both metal ions were equally effective in catalyzing the modification of alpha-crystallin.     

Energy transfer from tryptophan residues of proteins to 8-anilinonaphthalene-1-sulfonate

The binding of the apolar fluorescent dye 8-anilinonaphthalene-1-sulfonate (ANS) to bovine serum albumin (BSA), phospholipase A₂ (PLA₂), ovalbumin, lysozyme, cobrotoxin and N-acetyltryptophanamide was used to assess the factors affecting the efficiency of energy transfer from Trp residues to the ANS molecule. We found that the efficiency of energy transfer from Trp residues to ANS was associated with the ability of proteins to enhance the ANS fluorescence. At the same molar concentration of protein, BSA enhanced ANS fluorescence most among these proteins; its Trp fluorescence was drastically quenched by the addition of ANS. Fluorescence enhancement of ANS in PLA₂-ANS complex increased upon addition of Ca²⁺ or change of the buffer to acidicpH, resulting in a higher efficiency of energy transfer from Trp residues to ANS. There was limited ANS fluorescence enhancement with ovalbumin, lysozyme, cobrotoxin, and N-acetyltryptophanamide and a less efficient quenching in Trp fluorescence. The capabilities of proteins for binding with ANS correlated with the decrease in their Trp fluorescence being quenching by ANS. However, the microenvironment surrounding Trp residues of proteins did not affect the energy transfer. Based on these results, the factors that affected the energy transfer from Trp residues to ANS are discussed.     

A comparative study on intrinsic fluorescence of BSA and lysozyme proteins in presence of different divalent ions from their solution and thin film conformations

Optical emission behaviours of lysozyme and bovine serum albumin, from bulk and thin film geometry, were studied in the presence of three different divalent ions (Mg²⁺, Ca²⁺ or Ba²⁺) using different spectroscopic [steady‐state fluorescence, UV–Vis and Fourier transform infra‐red (FTIR)] techniques. Additionally, protein thin films on silicon surfaces were prepared and morphological studies were carried out using atomic force microscopy. Dynamic quenching was mainly identified for both proteins in the presence of Mg²⁺, Ca²⁺ and Ba²⁺ ions. The molecular conformation of the proteins was modified in thin films compared with that in solution, consequently quenching efficiencies also varied. ATR‐FTIR studies confirmed the conformational changes of proteins in the presence of all divalent ions. All metal ions used were divalent in nature and belonged to the same group of the periodic table but, depending on their individual characteristics such as electron affinity, ionic radius, etc., the magnitude of the protein and hydrated ion interaction varied and accordingly the quenching efficiency was modified. Quenching was maximum for Ca²⁺ ions, followed by the other two ions. Our study clearly illustrates the geometry‐dependent physical and biological functions of proteins.     

The Role of Histidine Residues in the Non-Enzyme Covalent Attachment of Glucose and Ascorbic Acid to Protein

Copper ions have been suggested to play a role in the non-covalent glycosylation (glycation) of proteins via transition metal-catalysed oxidations. We have further investigated “autoxidative glycosylation” by comparison of the behaviour of dog and bovine serum albumin with respect to the oxidative reactions of glucose and ascorbate. The proteins possess similar numbers of total amino residues available for glucose attachment but dog serum albumin contains fewer histidine groups and also lacks a high affinity copper-binding site. We find that the higher copper-binding capacity of bovine serum albumin is reflected in a lower rate of ascorbate oxidation as well as less protein oxidative damage than is the case for dog serum albumin. We also observe that modification of bovine serum albumin histidine groups by diethylpyrocarbonate enhances ascorbate-mediated protein fluorophore formation.     

Kinetics of superoxide dismutase- and iron-catalyzed nitration of phenolics by peroxynitrite.

Superoxide dismutase and Fe3+EDTA catalyzed the nitration by peroxynitrite (ONOO-) of a wide range of phenolics including tyrosine in proteins. Nitration was not mediated by a free radical mechanism because hydroxyl radical scavengers did not reduce either superoxide dismutase or Fe3+EDTA-catalyzed nitration and nitrogen dioxide was not a significant product from either catalyst. Rather, metal ions appear to catalyze the heterolytic cleavage of peroxynitrite to form a nitronium-like species (NO2+). The calculated energy for separating peroxynitrous acid into hydroxide ion and nitronium ion is 13 kcal.mol-1 at pH 7.0. Fe3+EDTA catalyzed nitration with an activation energy of 12 kcal.mol-1 at a rate of 5700 M-1.s-1 at 37 degrees C and pH 7.5. The reaction rate of peroxynitrite with bovine Cu,Zn superoxide dismutase was 10(5) M-1.s-1 at low superoxide dismutase concentrations, but the rate of nitration became independent of superoxide dismutase concentration above 10 microM with only 9% of added peroxynitrite yielding nitrophenol. We propose that peroxynitrite anion is more stable in the cis conformation, whereas only a higher energy species in the trans conformation can fit in the active site of Cu,Zn superoxide dismutase. At high superoxide dismutase concentrations, phenolic nitration may be limited by the rate of isomerization from the cis to trans conformations of peroxynitrite as well as by competing pathways for peroxynitrite decomposition. In contrast, Fe3+EDTA appears to react directly with the cis anion, resulting in greater nitration yields.     

Non-enzymatic glycation induces structural modifications of myoglobin

Increased glucose concentration in diabetes mellitus causes glycation of several proteins, leading to changes in their properties. Although glycation-induced functional modification of myoglobin is known, structural modification of the protein has not yet been reported. Here, we have studied glucose-modified structural changes of the heme protein. After in vitro glycation of metmyoglobin (Mb) by glucose at 25°C for 6 days, glycated myoglobin (GMb) and unchanged Mb have been separated by ion exchange (BioRex 70) chromatography, and their properties have been compared. Compared to Mb, GMb exhibits increased absorbance around 280 nm and enhanced fluorescence emission with excitation at 285 nm. Fluorescence quenching experiments of the proteins by acrylamide and KI indicate that more surface accessible tryptophan residues are exposed in GMb. CD spectroscopic study reveals a change in the secondary structure of GMb with decreased α-helix content. 1-anilino-naphthaline-8-sulfonate (ANS) binding with Mb and GMb indicates that glycation increases hydrophobicity of the heme protein. GMb appears to be less stable with respect to thermal denaturation and differential calorimetry experiments. Heme-globin linkage becomes weaker in GMb, as shown by spectroscopic and gel electrophoresis experiments. A correlation between glycation-induced structural and functional modifications of the heme protein has been suggested.     

Sensitivity of tryptophan and related compounds to oxidation induced by lipid autoperoxidation. Application to human serum low- and high-density lipoproteins

Tryptamine, serotonin and tryptophan are readily oxidized during the Cu2+-catalyzed peroxidation of arachidonic acid (AA) at neutral pH and under certain experimental conditions which determine their relative susceptibility to oxidation. Thus, in AA micelles, fluorescence spectroscopy demonstrates that positively-charged indoles interact with negatively-charged micelles while Trp remains in the aqueous phase. As a result, serotonin and tryptamine are preferentially oxidized. In egg phosphatidylcholine liposomes loaded with AA, the three substrates interact with vesicles and undergo lipid-induced oxidation. EDTA inhibits the formation of thiobarbituric-reactive substances (TBARS) and prevents the indoles from oxidation. Owing to the intricate contact between the lipidic core and the apolipoproteins, the Trp residues of human serum LDL and HDL3 are very rapidly oxidized, i.e., at least one order of magnitude faster than Tyr HDL and Lys LDL, which are believed to be involved in the binding of these lipoproteins to their cell receptors. Cupric ions are rather specific for the lipid-induced autoxidation of Trp residues of lipoproteins whereas in micelles and liposomes, Mn2+ and Fe2+ can lead to TBARS production and to oxidation of indoles. This specificity is surprising considering the known ability of Fe2+ to catalyze LDL modification (measured by TBARS production) during their incubation with various cells. Biological consequences of the easy lipid-induced oxidation of biologically important indoles are discussed.     

An in vitro study on the role of metal catalyzed oxidation in glyeation and crosslinking of collagen

The present investigation was carried out to understand the effect of metal catalyzed oxidation on glycation and crosslinking of collagen. Tail tendons obtained from rats weighing 200-225 g were incubated with glucose (250 mM) and increasing concentrations of copper ions (5, 25, 50 and 100 M) under physiological conditions of temperature and pH. Early glycation, crosslinking and late glycation (fluorescence) of collagen samples were analyzed periodically. Early glycation was estimated by phenol sulfuric acid method, and the crosslinking was assessed by pepsin and cyanogen bromide digestion. A concentrationdependent effect of metal ions on the rate of glycation and crosslinking of collagen was observed. Tendon collagen incubated with glucose and 100 M copper ions showed 80% reduction in pepsin digestion within seven days, indicating extensive crosslinking, whereas collagen incubated with glucose alone for the same period showed only 7% reduction. The presence of metal ions in the incubation medium accelerated the development of Maillard reaction fluorescence on collagen, and the increase was dependent on the concentration of metal ions used. The metal chelator Diethylene triamine penta-acetate significantly prevented the increase in collagen crosslinking by glucose and copper ions. Free radical scavengers benzoate and mannitol effectively prevented the increased crosslinking and browning of collagen by glucose. The results indicate that the metal catalyzed oxidation reactions play a major role in the crosslinking of collagen by glucose. It is also suggested that the prevention of increased oxidative stress in diabetes may prevent the accelerated advanced glycation and crosslinking of collagen.     

Speciation of trace elements in proteins in human and bovine serum by size exclusion chromatography and inductively coupled plasma-mass spectrometry with a magnetic sector mass spectrometer

 Proteins are separated by size exclusion chromatography while atomic ions from the inorganic elements are detected on-line by inductively coupled plasma-mass spectrometry. A double focusing mass analyzer provides very high sensitivity, low background, and sufficient spectral resolution to separate the atomic ions of interest from most polyatomic ions at the same nominal m/z value. The chromatograms show the distribution of the elements of interest between protein-bound and free fractions and provide the approximate molecular weights of those protein fractions that contain the elements monitored. The distribution of various elements, including V, Mo, Fe, Co, Mn, and lanthanides, in human or bovine serum samples are shown. Alkali metals and Tl are present primarily as free metal ions and are not bound to proteins. Inorganic elements spiked into the serum samples can be followed into various proteins. EDTA does not remove Fe, Pb, Sn, or Th from the proteins but does extract Mn from some proteins. Procedures for determining the effects of breaking disulfide linkages on the metal binding characteristics of proteins are also described. Received: 22 March 1999 / Accepted: 16 June 1999     

Changes in Ca2+ affinity upon activation of Agkistrodon piscivorus piscivorus phospholipase A2

Changes in the affinity of calcium for phospholipase A2 from Agkistrodon piscivorus piscivorus during activation of the enzyme on the surface of phosphatidylcholine vesicles have been investigated by site-directed mutagenesis and fluorescence spectroscopy. Changes in fluorescence that occur during lipid binding and subsequent activation have been ascribed to each of the three individual Trp residues in the protein. This was accomplished by generating a panel of mutant proteins, each of which lacks one or more Trp residues. Both Trp21, which is found in the interfacial binding region, and Trp119 show changes in fluorescence upon protein binding to small unilamellar zwitterionic vesicles or large unilamellar vesicles containing sufficient anionic lipid. Trp31, which is near the Ca2+ binding loop, exhibits little change in fluorescence upon lipid bilayer binding. A change in the fluorescence of the protein also occurs during activation of the enzyme. These changes arise from residue Trp31 as well as residues Trp21 and Trp119. The calcium dependence of the fluorescence change of Trp31 indicates that the affinity of the enzyme for calcium increases at least 3 orders of magnitude upon activation. These studies suggest either that a change in conformation of the enzyme occurs upon activation or that the increase in calcium affinity reflects formation of a ternary complex of calcium, enzyme, and substrate.     

Fragmentation of proteins by free radicals and its effect on their susceptibility to enzymic hydrolysis.

Defined radical species generated radiolytically were allowed to attack proteins in solution. The hydroxyl radical (OH.) in the presence of O2 degraded bovine serum albumin (BSA) to specific fragments detectable by SDS/polyacrylamide-gel electrophoresis; fragmentation was not obvious when the products were analysed by h.p.l.c. In the absence of O2 the OH. cross-linked the protein with bonds stable to SDS and reducing conditions. The superoxide (O2-.) and hydroperoxyl (HO2.) radicals were virtually inactive in these respects, as were several other peroxyl radicals. Fragmentation and cross-linking could also be observed when a mixture of biosynthetically labelled cellular proteins was used as substrate. Carbonyl and amino groups were generated during the reaction of OH. with BSA in the presence of O2. Changes in fluorescence during OH. attack in the absence of O2 revealed both loss of tryptophan and changes in conformation during OH. attack in the presence of O2. Increased susceptibility to enzymic proteolysis was observed when BSA was attacked by most radical systems, with the sole exception of O2-.. The transition-metal cations Cu2+ and Fe3+, in the presence of H2O2, could also fragment BSA. The reactions were inhibited by EDTA, or by desferal and diethylenetriaminepenta-acetic acid ('DETAPAC') respectively. The increased susceptibility to enzymic hydrolysis of radical-damaged proteins may have biological significance.     

Collagen in its fibrillar state is protected from glycation

To assess the impact collagen structures may have on glycation, the effects of glucose upon bovine serum albumin, guinea pig skin collagen, rat tail tendon and monomeric collagen were compared under near physiological conditions. Proteins were incubated with or without 50 mM glucose for 64 d in pH 7.4 50 mM phosphate buffer, followed by reduction, acid/alkaline hydrolysis, and analysis. Yields of non-reducible fructose-lysine, in the form of the acid-degradation products furosine and pyridosine, were significantly higher from skin collagen when compared to albumin. Yields of reducible fructose-lysine, in the form of glucitol- and mannitol-lysine, were conversely much greater for albumin, while tail tendon reported intermediate values. Fructose-lysine and unmodified lysine within collagen fibres prior to incubation was therefore protected by the tight packing of the collagen helices, where milling of tail tendon to increase the surface area exposed much of it to reduction protocols. Together with an analysis of pentosidine formation and other products, these results have shown that the interior of the tightly packed skin collagen fibres is protected from both glycation and reduction, and that glycation products differ depending on the protein incubated. Amino acid analysis then showed that our glycated skin collagen was similar to human diabetic skin collagen. Significant quantities of glucose-independent unknowns form in control incubations; their composition again being protein-dependent. The four compound Ks as previously reported were found to be unique to glycated rat tail tendon and soluble collagen, while another glycation product detected in collagen but not albumin may be attributable to carboxymethyl-arginine.     

Ascorbic acid, metal ions and the superoxide radical.

1. No evidence could be found for production of the superoxide radical, O2-, during autoxidation of ascorbic acid at alkaline pH values. Indeed, ascorbate may be important in protection against O2- genat-d in vivo. 2. Oxidation of ascorbate at pH 10.2 was stimulated by metal ions. Stimulation by Fe2+ was abolished by superoxide dismutase, probably because of generation of O2-- during reduction of O2 by Fe2+, followed by reaction of O2-- with ascorbate. EDTA changed the mechanism of Fe2+-stimulated ascorbate oxidation. 3. Stimulation of ascorbate oxidation by Cu2+ was also decreased by superoxide dismutase, but this appears to be an artifact, since apoenzyme or bovine serum albumin showed similar effects.     

Static and dynamic quenching of protein fluorescence. I. Bovine serum albumin.

The fluorescence parameters, lifetime, relative quantum yield, maximum and mean wavelength, half-width, and polarization, of bovine serum albumin (BSA) were measured at 15°C in aqueous solutions containing varying concentrations of different chemical perturbants, glycerol, Cu²⁺ ions, guanidine hydrochloride, and urea. By considering a quenching mechanism as being either dynamic or static, depending upon whether the quenching is or is not accompanied by a change in the fluorescence lifetime, we were able to correlate the changes produced in the various fluorescence parameters by the different chemical perturbants with changes in macromolecular structure as the concentration of perturbant was gradually increased. The addition of glycerol and of Cu²⁺ ions indicated that in aqueous BSA both tryptophan residues are below the surface of the macromolecule, out of contact with solvent water, and, as a consequence, they are statically quenched. “Ultra-Pure” guanidine hydrochloride at 2.4 M or more caused a drastic conformation change, which resulted in the emergence of a visible tyrosine peak at 304 nm in the BSA fluorescence spectrum when either 260- or 270-nm excitation was employed. With the same excitation, the enhancement of BSA tyrosine fluorescence by 6–8 M ultra-pure urea produced only a shoulder near 304 nm in the BSA fluorescence spectrum. We have introduced the use of a new relative quantum yield for protein fluorescence, q′, referenced to the quantum yield of unquenched free tryptophan, which eliminates the quenching action of water from the reference.     

Ribosylation of bovine serum albumin induces ROS accumulation and cell death in cancer line (MCF-7)

Formation of advanced glycation end products (AGE) is crucially involved in the several pathophysiologies associated with ageing and diabetes, for example arthritis, atherosclerosis, chronic renal insufficiency, Alzheimer’s disease, nephropathy, neuropathy, and cataracts. Because of devastating effects of AGE and the significance of bovine serum albumin (BSA) as a transport protein, this study was designed to investigate glycation-induced structural modifications in BSA and their functional consequences in breast cancer cell line (MCF-7). We incubated d-ribose with BSA and monitored formation of d-ribose-glycated BSA by observing changes in the intensity of fluorescence at 410 nm. NBT (nitro blue tetrazolium) assay was performed to confirm formation of keto-amine during glycation. Absorbance at 540 nm (fructosamine) increased markedly with time. Furthermore, intrinsic protein and 8-anilino-1-naphthalenesulfonate (ANS) fluorescence revealed marked conformational changes in BSA upon ribosylation. In addition, a fluorescence assay with thioflavin T (ThT) revealed a remarkable increase in fluorescence at 485 nm in the presence of glycated BSA. This suggests that glycation with d-ribose induced aggregation of BSA into amyloid-like deposits. Circular dichroism (CD) study of native and ribosylated BSA revealed molten globule formation in the glycation pathway of BSA. Functional consequences of ribosylated BSA on cancer cell line, MCF-7 was studied by MTT assay and ROS estimation. The results revealed cytotoxicity of ribosylated BSA on MCF-7 cells.     

Production and characterization of antibodies to advanced glycation products on proteins

Antibodies directed against advanced glycation products formed during Maillard reaction have been generated and characterized. These antibodies reacted specifically with advanced glycation products in common among proteins incubated with glucose, but not early-stage compounds such as a Schiff base adduct and Amadori rearrangement products. Incubation of bovine serum albumin with glucose caused a time-related increase in immunoreactivity and a concomitant increase in fluorescence intensity. These antibodies may serve as a useful tool to elucidate pathophysiological roles of advanced Maillard reaction in diabetic complications and aging processes.     

Detection of an advanced glycosylation product bound to protein in situ

Protein amino groups can react with glucose without the aid of enzymes to form stable Amadori products containing 1-amino-1-deoxyketose residues. These adducts can undergo subsequent rearrangements and dehydrations to form various brown and fluorescent pigments. Recently, a chromophore, 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI), was isolated from acid hydrolysates of bovine serum albumin (BSA) and poly-L-lysine which had been incubated with glucose. To confirm the presence of FFI in situ, a radioimmunoassay was developed. A derivative of FFI, 4-furanyl-2-furoyl-1H-imidazole-1-hexanoic acid, was coupled to BSA and used to immunize rabbits. A radioactive FFI derivative was synthesized by reaction of 2-furyl-glyoxal with gamma-amino-[2,3-3H]butyric acid to form FFI-[3H]butyric acid. The resultant antiserum showed binding affinity to FFI and cross-reactivity for related compounds. FFI bound to proteins was liberated by acid hydrolysis or digestion by proteinase K prior to measurement. A linear relationship was seen between the amount of FFI equivalent detected and the amount of acid hydrolysate or enzymatic digest assayed. Poly-L-lysine and BSA incubated with glucose showed a time-dependent increase in the amounts of fluorescence and FFI equivalence. The detection of a time-related increase in the amount of FFI or a closely related structure in enzymatically digested proteins implicates it as an in situ product on proteins which have undergone the Maillard reaction with glucose. Of physiological significance is that FFI could also be detected in human globin and serum albumin from normal individuals. Thus, proteins exposed to glucose in vitro and in vivo form FFI as an in situ glycosylation product.     

Photo-induced riboflavin binding to the tryptophan residues of bovine and human serum albumins

Summary The photobinding between riboflavin and the Trp residues from human and bovine serum albumins at two pH-dependent protein conformations was studied. At pH 7.0 both proteins showed photo-adduct formation with hyperbolic kinetics. In the bovine serum albumin this is attributed to the different locations of the two Trp residues. In the case of the human serum albumin, which has only one Trp residue, this behaviour may be related to different molecular conformations of the protein, as is also manifest in the iodide quenching experiments. At pH 3.5, the kinetics of the photo-adduct formation were found to be slower and showed a monophasic behaviour. These results are due to the conformational change of these proteins at acidic pH; the Trp residues of both proteins being now located in a more hydrophobic environment. When bovine serum albumin was anaerobically irradiated at pH 7.0 in the presence of¹⁴C-riboflavin and then cleaved by CNBr, two peptides were obtained, containing the Trp-134 and Trp-212 residues, respectively. The incorporation of¹⁴C-riboflavin in these samples was significantly higher at the level of the peptide containing the Trp-134 residue. Furthermore, it was demonstrated, that the energy transfer from enzymatically generated triplet acetone to riboflavin can also promote the binding of this vitamin to the Trp residues of human and bovine serum albumins.     

Direct determination of tryptophan using high-performance anion-exchange chromatography with integrated pulsed amperometric detection

Here we present a new method to rapidly quantify tryptophan (Trp) in proteins, animal feed (Mehaden fishmeal), cell cultures, and fermentation broths. Trp is separated from common amino acids by anion-exchange chromatography in 12min and directly detected by integrated pulsed amperometry. The estimated lower detection limit for this method is 1pmol. Alkaline (4M NaOH) hydrolysates can be directly injected, and therefore we used this method to determine the optimum alkaline hydrolysis conditions for the release of Trp from a model protein, bovine serum albumin (BSA). This method accurately determined the Trp content of BSA and fishmeal. High levels of glucose (2%, w/w) do not interfere with the chromatography or decrease recovery of Trp. We used this method to monitor free Trp during an Escherichia coli fermentation.     

A study of strontium binding to albumins, by a chromatographic method involving atomic emission spectrometric detection

A chromatographic method involving ICP-AES (inductively coupled plasma atomic emission spectrometry) detection has been successfully applied for the study of strontium-protein complexes. The chromatographic step involves the use of gel filtration-a large-zone Hummel and Dreyer method-which allows to dissociate the bound metallic ions and the free ones. This step is followed by an ICP-AES analysis of fractions collected throughout the chromatographic experiment: the concentration of ionic metallic species in solution can therefore be calculated. Two proteins have been tested: bovine serum albumin, which showed only weak interactions with Sr2+ ions, and bovine alpha-lactalbumin: this protein, well-known for its calcium binding capacity, proved to interact strongly with strontium. The influence of various parameters on the formation of strontium-lactalbumin complexes were determined, namely temperature, pH. Competition experiments between Sr2+ ions and, respectively Na+ and Ca2+ ions were also performed, by varying ionic strength of the medium, and by using both apo and native forms of bovine alpha-lactalbumin.