Effects of intermolecular thiol-disulfide interchange reactions on bsa fouling during microfiltration

Several studies have shown that one of the critical factors governing protein fouling of microfiltration membranes is the presence of denaturedand/or aggregated protein in the bulk solutions. Experiments were performed to evaluate the role of intermolecular disulfide interchange reactionson protein aggregation and membrane fouling during stirred cell microfiltration of bovine serum albumin (BSA). The flux decline during BSA filtration was quite dramatic due to the formation of a protein deposit thatfully covered the membrane pores. This flux decline could be completely eliminated by capping the free sulfhydryl group present on the BSA with eithera carboxymethyl or cysteinyl group, demonstrating the critical importance of this free thiol in the intermolecular aggregation reactions and, in turn, protein fouling. BSA aggregation during storage could be reduced by the addition of metal chelators (EDTA and citrate) or dithiothreitol, orby storage at lower pH (7.0) these solutions all had a significantly lower rate of fouling upon subsequent filtration. This behavior is completely consistent with the known chemistry of the thiol-disulfide interchange reaction, demonstrating that an understanding of these intermolecular (aggregation) reactions can provide a rational framework for the analysis and control of protein fouling in these membrane systems. (c) 1994 John Wiley & Sons, Inc.     

Non-native aggregation of recombinant human granulocyte-colony stimulating factor under simulated process stress conditions

Effective inhibition of protein aggregation is a major goal in biopharmaceutical production processes optimized for product quality. To examine the characteristics of process-stress-dependent aggregation of human granulocyte colony-stimulating factor (G-CSF), we applied controlled stirring and bubble aeration to a recombinant non-glycosylated preparation of the protein produced in Escherichia coli. We characterized the resulting denaturation in a time-resolved manner using probes for G-CSF conformation and size in both solution and the precipitate. G-CSF was precipitated rapidly from solutions that were aerated or stirred; only small amounts of soluble aggregates were found. Exposed hydrophobic surfaces were a characteristic of both soluble and insoluble G-CSF aggregates. Using confocal laser scanning microscopy, the aggregates presented mainly a circular shape. Their size varied according to incubation time and stress applied. The native intramolecular disulfide bonds in the insoluble G-CSF aggregates were largely disrupted as shown by mass spectrometry. New disulfide bonds formed during aggregation. All involved Cys(18) , which is the only free cysteine in G-CSF; one of them had an intermolecular Cys(18(A)) -Cys(18(B)) crosslink. Stabilization strategies can involve external addition of thiols and extensive reduction of surface exposition during processing.     

The active site cysteine of the proapoptotic protein glyceraldehyde-3-phosphate dehydrogenase is essential in oxidative stress-induced aggregation and cell death

Recent studies have revealed that the redox-sensitive glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is involved in neuronal cell death that is triggered by oxidative stress. GAPDH is locally deposited in disulfide-bonded aggregates at lesion sites in certain neurodegenerative diseases. In this study, we investigated the molecular mechanism that underlies oxidative stress-induced aggregation of GAPDH and the relationship between structural abnormalities in GAPDH and cell death. Under nonreducing in vitro conditions, oxidants induced oligomerization and insoluble aggregation of GAPDH via the formation of intermolecular disulfide bonds. Because GAPDH has four cysteine residues, including the active site Cys(149), we prepared the cysteine-substituted mutants C149S, C153S, C244A, C281S, and C149S/C281S to identify which is responsible for disulfide-bonded aggregation. Whereas the aggregation levels of C281S were reduced compared with the wild-type enzyme, neither C149S nor C149S/C281S aggregated, suggesting that the active site cysteine plays an essential role. Oxidants also caused conformational changes in GAPDH concomitant with an increase in beta-sheet content; these abnormal conformations specifically led to amyloid-like fibril formation via disulfide bonds, including Cys(149). Additionally, continuous exposure of GAPDH-overexpressing HeLa cells to oxidants produced disulfide bonds in GAPDH leading to both detergent-insoluble and thioflavin-S-positive aggregates, which were associated with oxidative stress-induced cell death. Thus, oxidative stresses induce amyloid-like aggregation of GAPDH via aberrant disulfide bonds of the active site cysteine, and the formation of such abnormal aggregates promotes cell death.     

Nanofibers made of globular proteins

Strong nanofibers composed entirely of a model globular protein, namely, bovine serum albumin (BSA), were produced by electrospinning directly from a BSA solution without the use of chemical cross-linkers. Control of the spinnability and the mechanical properties of the produced nanofibers was achieved by manipulating the protein conformation, protein aggregation, and intra/intermolecular disulfide bonds exchange. In this manner, a low-viscosity globular protein solution could be modified into a polymer-like spinnable solution and easily spun into fibers whose mechanical properties were as good as those of natural fibers made of fibrous protein. We demonstrate here that newly formed disulfide bonds (intra/intermolecular) have a dominant role in both the formation of the nanofibers and in providing them with superior mechanical properties. Our approach to engineer proteins into biocompatible fibrous structures may be used in a wide range of biomedical applications such as suturing, wound dressing, and wound closure.     

The secondary structure of pressure- and temperature-induced aggregates of equine serum albumin studied by FT-IR spectroscopy

The protein aggregation is divided into amyloid fibrils and amorphous aggregates. Amyloid fibrils are composed of the 3-dimensional ordered structure and are bound to thioflavin T and Congo red dyes. The amorphous aggregates with the disordered structure do not bind to these dyes. We have investigated the pressure- and heat-induced aggregates of equine serum albumin (ESA) from the secondary structural viewpoint using FT-IR spectroscopy. We show the secondary structural differences between heat- and pressure-induced aggregates of ESA. The heat-induced irreversible aggregates of ESA are composed of the intermolecular beta-sheet structure without binding thioflavie T and Congo red to be amorphous form. On the other hand, the pressure-induced reversible aggregates are composed of the random structure to be also amorphous form. From the comparison of pressure effects on ESA in native and reducing conditions of disulfide bridges, we demonstrate that the restriction of structural flexibility by disulfide bridges is an important factor for the reversibility of the pressure-induced aggregation.     

Epidermal fucosylation of cell surface glycoprotein

The formation of covalently linked, high molecular weight protein aggregates has been thought to play an important role in opacification of the human lens. Antisera were used in Western blot analysis to demonstrate the involvement of all major classes of lens proteins (alpha, beta and gamma crystallin; the major intrinsic membrane polypeptide) in covalent aggregation. Of these classes, aggregation of gamma and beta crystallins via intermolecular disulfide bonding and aggregation of the major intrinsic membrane polypeptide via intermolecular, non-disulfide bonding were more pronounced in cataractous as compared with normal lenses.     

Using intramolecular disulfide bonds in tau protein to deduce structural features of aggregation-resistant conformations

Because tau aggregation likely plays a role in a number of neurodegenerative diseases, understanding the processes that affect tau aggregation is of considerable importance. One factor that has been shown to influence the aggregation propensity is the oxidation state of the protein itself. Tau protein, which contains two naturally occurring cysteine residues, can form both intermolecular disulfide bonds and intramolecular disulfide bonds. Several studies suggest that intermolecular disulfide bonds can promote tau aggregation in vitro. By contrast, although there are data to suggest that intramolecular disulfide bond formation retards tau aggregation in vitro, the precise mechanism underlying this observation remains unclear. While it has been hypothesized that a single intramolecular disulfide bond in tau leads to compact conformations that cannot form extended structure consistent with tau fibrils, there are few data to support this conjecture. In the present study we generate oxidized forms of the truncation mutant, K18, which contains all four microtubule binding repeats, and isolate the monomeric fraction, which corresponds to K18 monomers that have a single intramolecular disulfide bond. We study the aggregation propensity of the oxidized monomeric fraction and relate these data to an atomistic model of the K18 unfolded ensemble. Our results argue that the main effect of intramolecular disulfide bond formation is to preferentially stabilize conformers within the unfolded ensemble that place the aggregation-prone tau subsequences, PHF6* and PHF6, in conformations that are inconsistent with the formation of cross-β-structure. These data further our understanding of the precise structural features that retard tau aggregation.     

Membrane fouling in sterile filtration of recombinant human growth hormone

The most troublesome problem encountered during the sterile filtration of protein solutions is membrane fouling. This article presents our study on sterile filtration of a model protein, recombinant human growth hormone (rhGH). Scanning electron microscopy (SEM) analysis shows that 0.22-mum membranes, when used to filter the mannitol-formulated protein solution under a 0.35-bar transmembrane pressure, were plugged to a great extent. When zinc ions were added to induce aggregates, the fouling tendency of rhGH solutions increased with increasing amount and size of the aggregates, indicating that the aggregates present before filtration might be responsible for membrane fouling. However, repeated filtration of the same solution using a fresh filter each time cannot reduce membrane fouling, and all filtrates contain the same trace amount of hGH particulates as the prefiltered solution. Particulate size was determined to be between 0.03 and 0.15 mum by dynamic light scattering. Also, in view of the fact that protein formulations significantly affected the tendency of fouling with the same preexisting aggregates, it is likely that fouling was more attributed to the aggregation taking place in the filter pores during filtration (secondary aggregation) than to the aggregates present before filtration. Adding a surfactant to or increasing the pH of the protein solution improves the filtration, whereas increasing ionic strength slows down the filtration. This result suggests that the balance of the protein's interaction and electrostatic repulsion plays an important role in the protein's fouling tendency. Many factors might change the microenvironment in the pores and disturb this balance. Those considerations and the aggregation tendency of rhGH in the filter pores will be discussed in detail separately. (c) 1996 John Wiley & Sons, Inc.     

Altered Thiol Chemistry in Human Amyotrophic Lateral Sclerosis-linked Mutants of Superoxide Dismutase 1

Neurodegenerative diseases share a common characteristic, the presence of intracellular or extracellular deposits of protein aggregates in nervous tissues. Amyotrophic Lateral Sclerosis (ALS) is a severe and fatal neurodegenerative disorder, which affects preferentially motoneurons. Changes in the redox state of superoxide dismutase 1 (SOD1) are associated with the onset and development of familial forms of ALS. In human SOD1 (hSOD1), a conserved disulfide bond and two free cysteine residues can engage in anomalous thiol/disulfide exchange resulting in non-native disulfides, a hallmark of ALS that is related to protein misfolding and aggregation. Because of the many competing reaction pathways, traditional bulk techniques fall short at quantifying individual thiol/disulfide exchange reactions. Here, we adapt recently developed single-bond chemistry techniques to study individual disulfide isomerization reactions in hSOD1. Mechanical unfolding of hSOD1 leads to the formation of a polypeptide loop held by the disulfide. This loop behaves as a molecular jump rope that brings reactive Cys-111 close to the disulfide. Using force-clamp spectroscopy, we monitor nucleophilic attack of Cys-111 at either sulfur of the disulfide and determine the selectivity of the reaction. Disease-causing mutations G93A and A4V show greatly altered reactivity patterns, which may contribute to the progression of familial ALS.     

Cysteine 111 affects aggregation and cytotoxicity of mutant Cu,Zn-superoxide dismutase associated with familial amyotrophic lateral sclerosis

Converging evidence indicates that aberrant aggregation of mutant Cu,Zn-superoxide dismutase (mutSOD1) is strongly implicated in familial amyotrophic lateral sclerosis (FALS). MutSOD1 forms high molecular weight oligomers, which disappear under reducing conditions, both in neural tissues of FALS transgenic mice and in transfected cultured cells, indicating a role for aberrant intermolecular disulfide cross-linking in the oligomerization and aggregation process. To study the contribution of specific cysteines in the mechanism of aggregation, we mutated human SOD1 in each of its four cysteine residues and, using a cell transfection assay, analyzed the solubility and aggregation of those SOD1s. Our results suggest that the formation of mutSOD1 aggregates are the consequence of covalent disulfide cross-linking and non-covalent interactions. In particular, we found that the removal of Cys-111 strongly reduces the ability of a range of different FALS-associated mutSOD1s to form aggregates and impair cell viability in cultured NSC-34 cells. Moreover, the removal of Cys-111 impairs the ability of mutSOD1s to form disulfide cross-linking. Treatments that deplete the cellular pool of GSH exacerbate mutSOD1s insolubility, whereas an overload of intracellular GSH or overexpression of glutaredoxin-1, which specifically catalyzes the reduction of protein-SSG-mixed disulfides, significantly rescues mutSOD1s solubility. These data are consistent with the view that the redox environment influences the oligomerization/aggregation pathway of mutSOD1 and point to Cys-111 as a key mediator of this process.     

Increased aggregation propensity of IgG2 subclass over IgG1: Role of conformational changes and covalent character in isolated aggregates

Aggregation of human therapeutic antibodies represents a significant hurdle to product development. In a test across multiple antibodies, it was observed that IgG1 antibodies aggregated less, on average, than IgG2 antibodies under physiological pH and mildly elevated temperature. This phenomenon was also observed for IgG1 and IgG2 subclasses of anti‐streptavidin, which shared 95% sequence identity but varied in interchain disulfide connectivity. To investigate the structural and covalent changes associated with greater aggregation in IgG2 subclasses, soluble aggregates from the two forms of anti‐streptavidin were isolated and characterized. Sedimentation velocity analytical ultracentrifugation (SV‐AUC) measurements confirmed that the aggregates were present in solution, and revealed that the IgG1 aggregate was composed of a predominant species, whereas the IgG2 aggregate was heterogeneous. Tertiary structural changes accompanied antibody aggregation as evidenced by greater ANS (8‐Anilino‐1‐naphthalene sulfonic acid) binding to the aggregates over monomer, and differences in disulfide character and tryptophan environments between monomer, oligomer and aggregate species, as observed by near‐UV circular dichroism (CD). Differences between subclasses were observed in the secondary structural changes that accompanied aggregation, particularly in the intermolecular β‐sheet and turn structures between the monomer and aggregate species. Free thiol determination showed ～2.4‐fold lower quantity of free cysteines in the IgG1 subclass, consistent with the 2.4‐fold reduction in aggregation of the IgG1 form when compared with IgG2 under these conditions. These observations suggested an important role for disulfide bond formation, as well as secondary and tertiary structural transitions, during antibody aggregation. Such degradations may be minimized using appropriate formulation conditions.     

Aggregation of recombinant bovine granulocyte colony stimulating factor in solution

Aggregation of recombinant bovine granulocyte colony-stimulating factor (rbG-CSF) was examined by the techniques of size exclusion chromatography (SEC), multiangle laser light scattering (MALS), and SDS-PAGE. Solutions of rbG-CSF in different buffers and pH were exposed to an elevated temperature of 50°C to induce aggregation. The formation of noncovalent soluble aggregates with molecular weight in the millions of Daltons was observed when a solution of rbG-CSF at pH 2.9 was exposed to 50°C. Precipitated protein was the main product of rbG-CSF aggregation in citrate and phosphate buffers at a pH greater than 4. It was demonstrated that precipitant was a mixture of covalent and noncovalent aggregates. The ratio of covalent to noncovalent binding increased with increase in pH of the protein solution. The covalent binding that occurred was primarily due to disulfide linkages via intermolecular disulfide scrambling as demonstrated by SDS-PAGE.     

Initial steps in assembly of microfibrils. Formation of disulfide-cross-linked multimers containing fibrillin-1

Fibrillins are the major constituents of extracellular microfibrils. How fibrillin molecules assemble into microfibrils is not known. Sequential extractions and pulse-chase labeling of organ cultures of embryonic chick aortae revealed rapid formation of disulfide-cross-linked aggregates containing fibrillin-1. These results demonstrated that intermolecular disulfide bond formation is an initial step in the assembly process. To identify free cysteine residues available for intermolecular cross-linking, small recombinant peptides of fibrillin-1 harboring candidate cysteine residues were analyzed. Results revealed that the first four cysteine residues in the unique N terminus form intramolecular disulfide bonds. One cysteine residue (Cys(204)) in the first hybrid domain of fibrillin-1 was found to occur as a free thiol and is therefore a good candidate for intermolecular disulfide bonding in initial steps of the assembly process. Furthermore, evidence indicated that the comparable cysteine residue in fibrillin-2 (Cys(233)) also occurs as a free thiol. These free cysteine residues in fibrillins are readily available for intermolecular disulfide bond formation, as determined by reaction with Ellman's reagent. In addition to these major results, the cleavage site of the fibrillin-1 signal peptide and the N-terminal sequence of monomeric authentic fibrillin-1 from conditioned fibroblast medium were determined.     

Intermolecular disulfide bond formation promotes immunoglobulin aggregation: Investigation by fluorescence correlation spectroscopy

Protein aggregation generally results from association between hydrophobic regions of individual monomers. However, additional mechanisms arising from specific interactions, such as intermolecular disulfide bond formation, may also contribute to the process. The latter is proposed to be the initiating pathway for aggregation of immunoglobulin (IgG), which is essential for triggering its immune response. To test the veracity of this hypothesis, we have employed fluorescence correlation spectroscopy to measure the kinetics of aggregation of IgG in separate experiments either allowing or inhibiting disulfide formation. Fluorescence correlation spectroscopy measurements yielded a diffusion time (τ_D) of ～200 µsec for Rhodamine‐labeled IgG, corresponding to a hydrodynamic radius (R_H) of 56 Å for the IgG monomer. The aggregation kinetics of the protein was followed by monitoring the time evolution of τ_D under conditions in which its cysteine residues were either free or blocked. In both cases, the progress curves confirmed that aggregation proceeded via the nucleation‐dependent polymerization pathway. However, for aggregation in the presence of free cysteines, the lag times were shorter, and the aggregate sizes bigger, than their respective counterparts for aggregation in the presence of blocked cysteines. This result clearly demonstrates that formation of intermolecular disulfide bonds represents a preferred pathway in the aggregation process of IgG. Fluorescence spectroscopy showed that aggregates formed in experiments where disulfide formation was prevented denatured at lower concentration of guanidine hydrochloride than those obtained in experiments where the disulfides were free to form, indicating that intermolecular disulfide bridging is a valid pathway for IgG aggregation. Proteins 2015; 83:169–177. © 2014 Wiley Periodicals, Inc.     

Mapping superoxide dismutase 1 domains of non-native interaction: roles of intra- and intermolecular disulfide bonding in aggregation

Superoxide dismutase 1 (SOD1) proteins harboring mutations linked to familial amyotrophic lateral sclerosis (FALS) uniformly show heightened potential to form high molecular weight structures. Here, we examine the domains of SOD1 that are involved in forming these structures (aggregates) and study the role of intra- and intermolecular disulfide bonds. An analysis of disease mutations identified to date reveals a non-random distribution with predominant occurrence at residues within highly conserved beta-strands or at highly conserved residues in loop domains. Using a cell transfection assay for aggregation, we determined that no single domain in SOD1 is indispensable in the formation of sedimentable aggregates, suggesting multiple potential motifs in the protein mediate non-native interactions. By a cell-free aggregation assay, analysis of transgenic mouse tissues, and mutagenesis approaches, we found evidence that redox conditions may modulate SOD1 aggregation; reduction of the native intramolecular disulfide bonds may predispose SOD1 to unfolding and aggregation, whereas non-native intermolecular disulfide linkages may help stabilize aggregates in vivo. The results suggest a possible mechanism for diversity in the structures formed by different SOD1 mutants, and define a potential contribution of redox conditions to SOD1 aggregation.     

Membrane fouling by cell-protein mixtures: in situ characterisation using multi-photon microscopy

Fouling of the membrane by cell and protein mixtures can result in severe flux declines, leading to the eventual need to clean or replace the membrane. In this study multi-photon microscopy, a fluorescence-based technique is used to 3-D image in situ the fouling of microfiltration membranes by suspensions containing combinations of washed yeast, bovine serum albumin (BSA) and ovalbumin. Appropriate fluorescent labelling allows the three foulant species to be clearly identified. Images correlate well with filtration data and clearly show the cake of yeast cells capturing protein aggregates. The proteins exhibited very different filtration behaviour. When filtering washed yeast together with ovalbumin and/or a 50:50 mixture by mass of BSA and ovalbumin, the ovalbumin fouling dominates the system. Capture of aggregates by the cake did not reduce fouling of the membrane by the protein and increased the resistance of the cake. For mixtures of BSA and washed yeast, the presence of a cake of yeast cells did reduce fouling of the membrane by the protein, however, the extra resistance due to the cake resulted in a flux lower than that when filtering BSA alone.     

Three-Dimensionally Printed Microfluidic Cross-flow System for Ultrafiltration/Nanofiltration Membrane Performance Testing

Minimization and management of membrane fouling is a formidable challenge in diverse industrial processes and other practices that utilize membrane technology. Understanding the fouling process could lead to optimization and higher efficiency of membrane based filtration. Here we show the design and fabrication of an automated three-dimensionally (3-D) printed microfluidic cross-flow filtration system that can test up to 4 membranes in parallel. The microfluidic cells were printed using multi-material photopolymer 3-D printing technology, which used a transparent hard polymer for the microfluidic cell body and incorporated a thin rubber-like polymer layer, which prevents leakages during operation. The performance of ultrafiltration (UF), and nanofiltration (NF) membranes were tested and membrane fouling could be observed with a model foulant bovine serum albumin (BSA). Feed solutions containing BSA showed flux decline of the membrane. This protocol may be extended to measure fouling or biofouling with many other organic, inorganic or microbial containing solutions. The microfluidic design is especially advantageous for testing materials that are costly or only available in small quantities, for example polysaccharides, proteins, or lipids due to the small surface area of the membrane being tested. This modular system may also be easily expanded for high throughput testing of membranes.      

Selective precipitation-assisted recovery of immunoglobulins from bovine serum using controlled-fouling crossflow membrane microfiltration

Efficient and economic recovery of immunoglobulins (Igs) from complex biological fluids such as serum, cell culture supernatant or fermentation cell lysate or supernatant, represents a substantial challenge in biotechnology. Methods such as protein A affinity chromatography and anion exchange chromatography are limited by cost and selectivity, respectively, while membrane chromatography is limited by low adsorptive area, flow distribution problems and scale-up difficulties. By combining the traditional salt-assisted precipitation process for selective removal of Igs from serum followed by constant-permeate flux membrane microfiltration for low fouling, we demonstrate an exciting new, efficient and economic hybrid method. The high selectivity of an ammonium sulfate-induced precipitation step was used to precipitate the Igs leaving the major undesirable impurity, the bovine serum albumin (BSA), in solution. Crossflow membrane microfiltration in diafiltration mode was then employed to retain the precipitate, while using axial flow rates to optimize removal of residual soluble BSA to the permeate. The selectivity between immunoglobulin G (IgG) and BSA obtained from the precipitation step was approximately 36, with 97% removal of the BSA with diafiltration in 5 diavolumes with resulting purity of the IgG of approximately 93% after the membrane microfiltration step. Complete resolubilization of the IgG was obtained without any aggregation at the concentrations of ammonium sulfate employed in this work. Further, membrane pore size and axial Reynolds number (recirculation rate) were shown to be important for minimizing fouling and loss of protein precipitate.     

Intermolecular cross-links mediate aggregation of phospholipid vesicles by pulmonary surfactant protein SP-A

As the most abundant glycoprotein component of pulmonary surfactant, SP-A (Mr = 30,000-36,000) plays a central role in the organization of phospholipid bilayers in the alveolar air space. SP-A, isolated from lung lavage, exists in oligomeric forms (N = 6, 12, 18, ...), mediated by collagen-like triple helices and intermolecular disulfide bonds. These protein-protein interactions, involving the amino-terminal domain of SP-A, are hypothesized to facilitate the alignment of surfactant lipid bilayers into unique tubular myelin structures. SP-A reorganization of surfactant lipid was assessed in vitro by quantitating the calcium-dependent light scattering properties of lipid vesicle suspensions induced by SP-A. Accelerated aggregation of unilamellar vesicles required SP-A and at least 3 mM free calcium. The initial rate of aggregation was proportional to the concentration of canine SP-A over lipid:protein molar ratios ranging from 200:1 to 5000:1. Digestion with bacterial collagenase or incubation with dithiothreitol (DTT) completely blocked lipid aggregation activity. Both treatments decreased the binding of SP-A to phospholipids. The conditions used in the DTT experiments (10 mM DTT, nondenaturing Tris buffer, 37 degrees C) resulted in the selective reduction and 14C-alkylation of the intermolecular disulfide bond involving residue 9Cys, whereas the four cysteines found in the noncollagenous domain of SP-A were inefficiently alkylated with [14C]-iodoacetate. HPLC analysis of tryptic SP-A peptides revealed that these four cysteine residues participate in intramolecular disulfide bond formation (138Cys-229Cys and 207Cys-221Cys). Our data demonstrate the importance of the quaternary structure (triple helix and intermolecular disulfide bond) of SP-A for the aggregation of unilamellar phospholipid vesicles.     

Fourier transform IR attenuated total reflectance spectroscopy studies of cysteine-induced changes in secondary conformations of bovine serum albumin after UV-B irradiation

Fourier transform IR spectroscopy equipped with attenuated total reflection was used to investigate the cysteine-induced alteration of the protein secondary structure of bovine serum albumin (BSA) in aqueous solution before and after UV-B irradiation. Several amino acids were also studied. The results indicate the unchanged IR spectra of BSA coincubated with amino acids, except cysteine, did not change after 72-h UV-B irradiation. There was no difference in the IR spectrum of the unirradiated BSA coincubated with cysteine. A shoulder at 1620 cm(-1) attributed to the intermolecular beta-sheet structure was observed for the IR spectrum of BSA coincubated with cysteine after 72-h UV-B irradiation. Moreover, the peak intensity at 1303 cm(-1) that is due the alpha-helix structure was reduced, but the peak intensity at 1247 cm(-1) corresponding to beta-sheet structures was increased. Longer UV-B exposure for a BSA solution coincubated with cysteine changed the BSA solution from clear to viscous to gel form in which a transparent gel and another white gel were simultaneously observed. A gradual IR spectral alteration was found for BSA coincubated with cysteine and subjected to increased UV-B irradiation. The longer UV-B irradiation yielded increased intensity at 1620 cm(-1). The second-derivative IR peaks at 1655, 1631, and 1548 cm(-1) were shifted to 1650, 1620, and 1544 cm(-1), respectively, by the increase of UV-B irradiation, suggesting a progressive transformation from an alpha-helix to an intermolecular beta-sheet structure for BSA coincubated with cysteine. This strongly implies that longer UV-B exposure time for the BSA solution in the presence of cysteine did alter the protein secondary structures of BSA more, thus inducing gel formation by protein aggregation.