Acid Denaturation Research Articles

Structural Characteristics of Extracted Collagen from Tilapia (Oreochromis mossambicus) Bone: Effects of Ethylenediaminetetraacetic Acid Solution and Hydrochloric Acid Treatment

Desalting is an important process of bone collagen extraction. In this present study, collagens were prepared from tilapia bone using acetic acid and pepsin. The extracted collagen was partially characterized. Prior to extraction, tilapia bone was desalted by soaking in ethylenediaminetetraacetic acid solution and hydrochloric acid. Thermo-gravimetric analysis, X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy were performed to analyze the structural characteristics. The extracted protein was classified as collagen by Fourier transform infrared spectroscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and circular dichroism. Results showed that desalination with HCl was fast, but collagen yield was low. No acid-soluble collagen and only 0.5% pepsin-soluble collagen were obtained from HCl-treated fish bone. By comparison, the yields of acid-soluble collagen and pepsin-soluble collagen from ethylenediaminetetraacetic acid-treated fish bone were 3.5 and 6.0%, respectively. These values were observed higher than those from HCl-treated fish bone. In HCl-treated fish bone, the properties of collagen, such as amino acid composition, denaturation temperature, and molecular weight distribution, were different from those of collagen in ethylenediaminetetraacetic acid-treated fish bone. Collagens extracted from ethylenediaminetetraacetic acid-treated fish bone showed more integrated secondary structure. Therefore, ethylenediaminetetraacetic acid can be used more effectively than HCl to extract collagen from tilapia bone.

Enteric-coated capsule containing β-galactosidase-loaded polylactic acid nanocapsules: enzyme stability and milk lactose hydrolysis under simulated gastrointestinal conditions

In order to protect peroral β-galactosidase from being degraded and hydrolyse milk lactose efficiently in the environments of gastrointestinal tract, a double-capsule delivery system composed of enteric-coated capsule and polylactic acid (PLA) nanocapsules (NCs) was developed for encapsulation of β-galactosidase. β-galactosidase-loaded PLA NCs in the size range of 100-200nm were prepared by a modified w1/o/w2 technique. During the encapsulation process, dichloromethane/ethyl acetate (1:1, v/v) as the solvent composition, high-pressure homogenisation (150 bar, 3min) as the second emulsification method and polyvinyl alcohol or Poloxamer 188 as a stabiliser in the inner phase could efficiently improve the activity retention of β-galactosidase (>90%). Subsequently, the prepared NCs were freeze-dried and filled in a hydroxypropyl methylcellulose phthalate (HP55)-coated capsule. In vitro results revealed that the HP55-coated capsule remained intact in the simulated gastric fluid and efficiently protected the nested β-galactosidase from acidic denaturation. Under the simulated intestinal condition, the enteric coating dissolved rapidly and released the β-galactosidase-loaded PLA NCs, which exhibited greater stability against enzymatic degradation and higher hydrolysis ratio (∼100%) towards milk lactose than the free β-galactosidase. These results suggest that this double-capsule delivery system represents promising candidate for efficient lactose hydrolysis in the gastrointestinal tract.

In silico single strand melting curve: a new approach to identify nucleic acid polymorphisms in Totiviridae.

BackgroundThe PCR technique and its variations have been increasingly used in the clinical laboratory and recent advances in this field generated new higher resolution techniques based on nucleic acid denaturation dynamics. The principle of these new molecular tools is based on the comparison of melting profiles, after denaturation of a DNA double strand. Until now, the secondary structure of single-stranded nucleic acids has not been exploited to develop identification systems based on PCR. To test the potential of single-strand RNA denaturation as a new alternative to detect specific nucleic acid variations, sequences from viruses of the Totiviridae family were compared using a new in silico melting curve approach. This family comprises double-stranded RNA virus, with a genome constituted by two ORFs, ORF1 and ORF2, which encodes the capsid/RNA binding proteins and an RNA-dependent RNA polymerase (RdRp), respectively.ResultsA phylogenetic tree based on RdRp amino acid sequences was constructed, and eight monophyletic groups were defined. Alignments of RdRp RNA sequences from each group were screened to identify RNA regions with conserved secondary structure. One region in the second half of ORF2 was identified and individually modeled using the RNAfold tool. Afterwards, each DNA or RNA sequence was denatured in silico using the softwares MELTSIM and RNAheat that generate melting curves considering the denaturation of a double stranded DNA and single stranded RNA, respectively. The same groups identified in the RdRp phylogenetic tree were retrieved by a clustering analysis of the melting curves data obtained from RNAheat. Moreover, the same approach was used to successfully discriminate different variants of Trichomonas vaginalis virus, which was not possible by the visual comparison of the double stranded melting curves generated by MELTSIM.ConclusionIn silico analysis indicate that ssRNA melting curves are more informative than dsDNA melting curves. Furthermore, conserved RNA structures may be determined from analysis of individuals that are phylogenetically related, and these regions may be used to support the reconstitution of their phylogenetic groups. These findings are a robust basis for the development of in vitro systems to ssRNA melting curves detection.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2105-15-243) contains supplementary material, which is available to authorized users.

Cataract-linked mutation R188H promotes βB2-crystallin aggregation and fibrillization during acid denaturation

Cataract is characterized by the formation of light-scattering protein aggregates in the lens. β/γ-Crystallins are the predominant structural proteins in the cytosol of lens fiber cells, and more than fifty β/γ-crystallin mutations have been linked to autosomal dominant congenital cataract. However, the structural role of these mutations in the formation of the core structures of amorphous aggregates or amyloid-like fibrils has not been elucidated yet. In this research, we studied the effects of the V187M and R188H mutations on the aggregation and fibrillization of βB2-crystallin during acid denaturation. The behavior of V187M was the same as the WT protein, suggesting that the residue at position 187 contributed little to the aggregation/fibrillization process. R188H promoted the formation of amorphous aggregates at pH above 3 and accelerated fibrillization at pH 3. The distinct behaviors of the mutants suggested that the residue at position 188 might play a regulatory role in βB2-crystallin aggregation/fibrillization but not reside in the core of the aggregates/fibrils.

Short-Chain Flavor Ester Synthesis in Organic Media by an E. coli Whole-Cell Biocatalyst Expressing a Newly Characterized Heterologous Lipase

Short-chain aliphatic esters are small volatile molecules that produce fruity and pleasant aromas and flavors. Most of these esters are artificially produced or extracted from natural sources at high cost. It is, however, possible to ‘naturally’ produce these molecules using biocatalysts such as lipases and esterases. A gene coding for a newly uncovered lipase was isolated from a previous metagenomic study and cloned into E. coli BL21 (DE3) for overexpression using the pET16b plasmid. Using this recombinant strain as a whole-cell biocatalyst, short chain esters were efficiently synthesized by transesterification and esterification reactions in organic media. The recombinant lipase (LipIAF5-2) showed good affinity toward glyceryl trioctanoate and the highest conversion yields were obtained for the transesterification of glyceryl triacetate with methanol. Using a simple cetyl-trimethylammonium bromide pretreatment increased the synthetic activity by a six-fold factor and the whole-cell biocatalyst showed the highest activity at 40°C with a relatively high water content of 10% (w/w). The whole-cell biocatalyst showed excellent tolerance to alcohol and short-chain fatty acid denaturation. Substrate affinity was equally effective with all primary alcohols tested as acyl acceptors, with a slight preference for methanol. The best transesterification conversion of 50 mmol glyceryl triacetate into isoamyl acetate (banana fragrance) provided near 100% yield after 24 hours using 10% biocatalyst loading (w/w) in a fluidized bed reactor, allowing recycling of the biocatalyst up to five times. These results show promising potential for an industrial approach aimed at the biosynthesis of short-chain esters, namely for natural flavor and fragrance production in micro-aqueous media.

Studies on the Mechanism of Electron Bifurcation Catalyzed by Electron Transferring Flavoprotein (Etf) and Butyryl-CoA Dehydrogenase (Bcd) of Acidaminococcus fermentans

Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the low-potential [4Fe-4S] clusters of ferredoxin, which increases the efficiency of the substrate level and electron transport phosphorylations. Here we characterize the bifurcating electron transferring flavoprotein (EtfAf) and butyryl-CoA dehydrogenase (BcdAf) of Acidaminococcus fermentans, which couple the exergonic reduction of crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. EtfAf contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Å. The EtfAf-NAD(+) complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH(-) is considered as the bifurcating electron donor. As a result of a domain movement, α-FAD is able to approach β-FADH(-) by about 4 Å and to take up one electron yielding a stable anionic semiquinone, α-FAD, which donates this electron further to Dh-FAD of BcdAf after a second domain movement. The remaining non-stabilized neutral semiquinone, β-FADH(•), immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and Dh-FADH(-) that converts crotonyl-CoA to butyryl-CoA.

Development and performance evaluation of a streamlined method for nucleic acid purification, denaturation, and multiplex detection of Bluetongue virus and Epizootic hemorrhagic disease virus

Bluetongue virus (BTV) and Epizootic hemorrhagic disease virus (EHDV) possess similar structural and molecular features, are transmitted by biting midges (genus Culicoides), and cause similar diseases in some susceptible ruminants. Generally, BTV causes subclinical disease in cattle, characterized by a prolonged viremia. EHDV-associated disease in cattle is less prominent; however, it has emerged as a major economic threat to the white-tailed deer (Odocoileus virginianus) industry in many areas of the United States. The recent emergence of multiple BTV and EHDV serotypes previously undetected in the United States demonstrates the need for robust detection of all known serotypes and differential diagnosis. For this purpose, a streamlined workflow consisting of an automated nucleic acid purification and denaturation method and a multiplex one-step reverse transcription quantitative polymerase chain reaction for the simultaneous detection of BTV serotypes 1-24 and EHDV serotypes 1-7 was developed using previously published BTV and EHDV assays. The denaturation of double-stranded (ds) BTV and EHDV RNA was incorporated into the automated nucleic acid purification process thus eliminating the commonly used separate step of dsRNA denaturation. The performance of this workflow was compared with the World Organization of Animal Health BTV reference laboratory (National Veterinary Services Laboratory, Ames, Iowa) workflow for BTV and EHDV detection, and high agreement was observed. Implementation of the workflow in routine diagnostic testing enables the detection of, and differentiation between, BTV and EHDV, and coinfections in bovine blood and cervine tissues, offering significant benefits in terms of differential disease diagnosis, herd health monitoring, and regulated testing.

Effects of Nanoscale Confinement on the Functionality of Nucleic Acids: Implications for Nanomedicine

The facile self-assembly and nanomanipulation of nucleic acids hold great promise in the design of innovative, programmable materials, with applications ranging from biosensing to cellular targeting and drug delivery. Little is known, however, of the effects of confinement on biochemical reactions within such systems, in which the level of packing and crowding is similar to that of intracellular environments. In this review article we outline novel, unexpected properties of nucleic acids that arise from nanoscale confinement, as mainly revealed by atomic force and electron microscopy, electrochemistry, fluorescence spectroscopy, and gel electrophoresis. We review selected scientific studies over the last decade that describe the novel behavior of nanoconfined nucleic acids with respect to hybridization, denaturation, conformation, stability, and enzyme accessibility. The nanoscale systems discussed include self-assembled, water-soluble, DNA or RNA nanostructures, ranging in width from a few to several tens of nm; gold nanoparticles coated with DNA monolayers; and self-assembled monolayers of DNA, from a few to several hundreds of bp in length. These studies reveal that the functionality of nucleic acid-based nanosystems is highly dependent upon the local density, molecular flexibility and network of weak interactions between adjacent molecules. These factors significantly affect steric hindrance, molecular crowding and hydration, which in turn control nucleic acid hybridization, denaturation, conformation, and enzyme accessibility. The findings discussed in this review article demonstrate that nucleic acids function in a qualitatively different manner within nanostructured systems, and suggest that these novel properties, if better understood, will enable the development of powerful molecular tools for nanomedicine.

Effect of Simulated Gastric and Intestinal Digestion on Temporal Stability and Immunoreactivity of Peanut, Almond, and Pine Nut Protein Allergens

Current models of digestibility utilize pepsin stability to assess the safety of allergenic versus nonallergenic food proteins. Dietary protein digestion in vivo, however, requires acid denaturation and protease cleavage by pepsin, trypsin, and/or chymotrypsin. The ability of this approach to identify food protein stability in the mammalian gut may be limited. We determined the temporal stability and immunoreactivity of almond, pine nut, and peanut allergenic proteins under simulated physiologic gastric and intestinal digestive conditions in vitro. Gel electrophoresis and immunoblot analyses were used to determine protein stability and immunoreactivity, respectively. Peanut, almond, and pine nut proteins were pepsin- and pancreatin-stable and immunoreactive for up to 1 h after initiation of digestion. Moreover, successive acid denaturation and pepsin and pancreatin cleavage were necessary to hydrolyze these allergenic proteins and reduce their IgG- and IgE-binding capacity, which suggests that digestibility models must be improved for more accurate safety assessment of food allergens.

The effect of simulated gastric and intestinal digestion on temporal stability and immunoreactivity of peanut, almond and pine nut protein allergens (P6206)

Abstract Current models of digestibility utilize pepsin stability to assess the safety of allergenic versus non-allergenic food proteins. Dietary protein digestion in vivo, however, requires acid denaturation and protease cleavage by pepsin, trypsin, and/or chymotrypsin. The ability of this approach to identify food protein stability in the mammalian gut may be limited. We determined the temporal stability and immunoreactivity of almond, pine nut, and peanut allergenic proteins using simulated physiologic gastric and intestinal digestive conditions in vitro. Gel electrophoresis and immunoblot analyses were used to determine protein stability and immunoreactivity, respectively. Peanut, almond, and pine nut proteins were pepsin- and pancreatin-stable, and immunoreactive for up to 1 h after initiating digestion. Moreover, successive acid denaturation, and pepsin and pancreatin cleavage were necessary to hydrolyze these allergenic proteins and reduce their IgG and IgE-binding capacity, which suggests that digestibility models must be improved for more accurate safety assessment of food allergens.

Monomeric Banana Lectin at Acidic pH Overrules Conformational Stability of Its Native Dimeric Form

Banana lectin (BL) is a homodimeric protein categorized among jacalin-related family of lectins. The effect of acidic pH was examined on conformational stability of BL by using circular dichroism, intrinsic fluorescence, 1-anilino-8-napthalene sulfonate (ANS) binding, size exclusion chromatography (SEC) and dynamic light scattering (DLS). During acid denaturation of BL, the monomerization of native dimeric protein was found at pH 2.0. The elution profile from SEC showed two different peaks (59.65 ml & 87.98 ml) at pH 2.0 while single peak (61.45 ml) at pH 7.4. The hydrodynamic radii (R h) of native BL was 2.9 nm while at pH 2.0 two species were found with R h of 1.7 and 3.7 nm. Furthermore at, pH 2.0 the secondary structures of BL remained unaltered while tertiary structure was significantly disrupted with the exposure of hydrophobic clusters confirming the existence of molten globule like state. The unfolding of BL with different subunit status was further evaluated by urea and temperature mediated denaturation to check their stability. As inferred from high Cm and ΔG values, the monomeric form of BL offers more resistance towards chemical denaturation than the native dimeric form. Besides, dimeric BL exhibited a Tm of 77°C while no loss in secondary structures was observed in monomers even up to 95°C. To the best of our knowledge, this is the first report on monomeric subunit of lectins showing more stability against denaturants than its native dimeric state.

Acid Denaturation and Refolding of Cytochrome c on Silica Surface

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Cis association of leukocyte Ig‐like receptor 1 with MHC class I modulates accessibility to antibodies and HCMV UL18

Leukocyte Ig-like receptor (LIR) 1 (CD85j/ILT2/LILRB1) is an inhibitory receptor with broad specificity for MHC class I (MHC-I) and the human CMV MHC-I homologue UL18. LIR-1 can inhibit NK cells through the conventional interaction with MHC-I expressed on a target cell (in trans) but the nature and the effects of LIR-1 interactions with MHC-I in cis are not well understood. Here we show that MHC-I expressed in cis has an impact on the detection of LIR-1 with various antibodies. We found the cis interaction alters recognition by only one of two antibodies known to block functional trans recognition by LIR-1 on NK cells. Specifically, we observed an enhancement of recognition with GHI/75 in the presence of various MHC-I alleles on 721.221 cells. We found that blocking the LIR-1 contact site with anti-MHC-I antibodies decreased detection of LIR-1 with GHI/75. We also observed a decrease in GHI/75 following acid denaturation of MHC-I. Finally, disruption of LIR-1 cis interactions with MHC-I significantly enhanced UL18-Fc binding to NK92 cells and enhanced the relative inhibition of NK92 cells by HLA-G. These results have implications for LIR-1 function in scenarios such as infection when MHC-I levels on effector cells may be increased by IFNs.

Different Conformation of Thiol Protease Inhibitor During Amyloid Formation: Inhibition by Curcumin and Quercetin

Cystatins are thiol proteinase inhibitors ubiquitously present in mammalian body and serve various important physiological functions. In the present study, we examined the effects of acid denaturation on newly identified thiol protease inhibitors from the lungs of Capra hircus (Goat) with a focus on protein conformational changes and amyloid fibril formation. Acid denaturation as studied by CD (Circular Dichroism) and fluorescence spectroscopy showed that purified inhibitor named GLC (Goat Lung Cystatin) populates three partly unfolded species, a native like state at pH 3.0, a partly unfolded intermediate at pH2.0, and unstructured unfolded state at pH 1.0, from each of which amyloid like fibrils grow as assessed by thioflavin T (ThT) spectroscopy. The result showed, native like structure formed at pH 3.0 is more responsive towards amyloid formation when compare to other conformation of proteins. Morphology of the protein species incubated for amyloid process was observed using transmission electron microscopy (TEM). Moreover, anti-fibrillogenic effects of curcumin and quercetin were analysed using ThT binding assay. Curcumin and quercetin produced a concentration dependent decline inThT fluorescence suggesting deaggregation of the fibrils. When added prior to amyloid fibril initiation 50 μM curcumin inhibited amyloid aggregation. However, more quercetin is needed to prevent the same extent of fibrillation. Implications for therapeutics in view of polyphenols as essential nutrients are suggested in lung diseases.

Определение аскорбиновой кислоты в крови методом ВЭЖХ

Sample preparation and HPLC conditions for the quantitative determination of ascorbic acid in objects with a complex matrix (blood) were studied. Influences of ascorbic acid degradation in the presence of different stabilizers and a sample matrix on the determination accuracy and sample stability were investigated. It was shown the possibility of ascorbic acid determination in blood samples, provided the mandatory acid denaturation in presence of the complexing and reducing agents. Data on stability of ascorbic acid in aqueous solutions and blood samples are provided. Key words: Ascorbic acid, HPLC, blood samples (Russian) DOI: http://dx.doi.org/10.15826/analitika.2013.17.3.008 V.V. Khasanov, K.A. Dychko, T.T. Kuriaeva, E.V. Nesterova Tomsk State University , Tomsk, Russian Federation

An optimized DNA extraction and purification method from dairy manure compost for genetic diversity analysis

An unbiased DNA extraction protocol is necessary for analysis of genetic diversity, particularly, of genes in complex environmental samples by nucleic acid techniques. In the present study, three manual extraction methods and two commonly used commercial kits, which were accompanied by two DNA purification strategies, were compared based on cell lysis efficiency, DNA and humic acid yields, PCR amplification and denaturing gradient gel electrophoresis (DGGE) analysis. The results show that in spite of higher cell lysis efficiencies of the two commercial kits, the purified DNA yields were only one-third of that obtained by the two manual methods of FTSP (Freeze-thaw-SDS-Protein K) and FTSPP (Freeze-thaw-SDS-Protein K-Polyvinylpolypyrrolidone). The purified DNA from all five methods was pure enough for successful PCR and real-time PCR amplifications in the presence of 1 μg μL(-1) BSA. However, the FTSPP extraction method with DNA purification by a Wizard(®) kit yielded the largest number of 16S rRNA gene copies and ribotypes or bands in DGGE profiles, which indicated a superiority over the other four methods. The development of this optimized DNA extraction and purification method may provide a valuable tool for further molecular analysis of compost.

Acid Denaturation and Refolding of Cytochrome c on Silica Surface

AbstractDenaturation of oxidized cytochrome c (cyt c) adsorbed to a hydrophilic fused silica surface was studied by UV‐VIS attenuated total reflection (ATR) spectroscopy using a multiple optical pass system newly developed by this lab. Cyt c surface adsorption at neutral pH gave an adsorption equilibrium constant of Ka = 2 × 105 M−1 and a surface coverage at 63% of a monolayer saturation. Protein unfolding by acid denaturation was studied by equilibrating surface bound cyt c with acid buffers ranging in pH from 5 to 2. Protein orientation and surface coverage were calculated based on a theoretical model developed in previous work. The average heme tilt angle (44°) was found to be independent of pH, implicating protein‐surface interactions as the dominant factor governing adsorption. A non‐random molecular orientation distribution of cyt c on the surface was observed, providing further support for the dominance of protein‐surface interactions. It was shown that when denaturing acid buffers were removed and replaced with a neutral buffer cyt c refolded, assuming their original conformation. The combination of unique, yet applicable, science and laboratory skills involved in this project had a tremendous impact on the authors‘ undergraduate curriculum, making it ideal for capstone project development.

Microfluidic-Based Amplification-Free Bacterial DNA Detection by Dielectrophoretic Concentration and Fluorescent Resonance Energy Transfer Assisted in Situ Hybridization (FRET-ISH).

Although real-time PCR (RT-PCR) has become a diagnostic standard for rapid identification of bacterial species, typical methods remain time-intensive due to sample preparation and amplification cycle times. The assay described in this work incorporates on-chip dielectrophoretic capture and concentration of bacterial cells, thermal lysis, cell permeabilization, and nucleic acid denaturation and fluorescence resonance energy transfer assisted in situ hybridization (FRET-ISH) species identification. Combining these techniques leverages the benefits of all of them, allowing identification to be accomplished completely on chip less than thirty minutes after receipt of sample, compared to multiple hours required by traditional RT-PCR and its requisite sample preparation.

Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization

The kinesin-13 Kif2C hydrolyzes ATP and uses the energy released to disassemble microtubules. The mechanism by which this is achieved remains elusive. Here we show that Kif2C-(sN+M), a monomeric construct consisting of the motor domain with the proximal part of the N-terminal Neck extension but devoid of its more distal, unstructured, and highly basic part, has a robust depolymerase activity. When detached from microtubules, the Kif2C-(sN+M) nucleotide-binding site is occupied by ATP at physiological concentrations of adenine nucleotides. As a consequence, Kif2C-(sN+M) starts its interaction with microtubules in that state, which differentiates kinesin-13s from motile kinesins. Moreover, in this ATP-bound conformational state, Kif2C-(sN+M) has a higher affinity for soluble tubulin compared with microtubules. We propose a mechanism in which, in the first step, the specificity of ATP-bound Kif2C for soluble tubulin causes it to stabilize a curved conformation of tubulin heterodimers at the ends of microtubules. Data from an ATPase-deficient Kif2C mutant suggest that, then, ATP hydrolysis precedes and is required for tubulin release to take place. Finally, comparison with Kif2C-Motor indicates that the binding specificity for curved tubulin and, accordingly, the microtubule depolymerase activity are conferred to the motor domain by its N-terminal Neck extension.

Thiol-Based Photocycle of the Blue and Teal Light-Sensing Cyanobacteriochrome Tlr1999

Cyanobacteriochromes are a spectrally diverse photoreceptor family that binds a bilin chromophore. For some cyanobacteriochromes, in addition to the widely conserved cysteine to anchor the chromophore, its ligation with a second cysteine is responsible for a remarkable blue shift. Herein, we report a newly discovered cyanobacteriochrome Tlr1999 exhibiting reversible photoconversion between a blue-absorbing form at 418 nm (P418) and a teal-absorbing form at 498 nm (P498). Acidic denaturation suggests that P418 harbors C15-Z phycoviolobilin, whereas P498 harbors C15-E phycoviolobilin. When treated with iodoacetamide, which irreversibly modifies thiol groups, P418 is slowly converted to a green-absorbing photoinactive form denoted P552. The absorption spectrum of P498 appears to be unaffected by iodoacetamide, but when iodoacetamide modified, it is photoconverted to P552. These results suggest that a covalent bond exists between the second Cys and the phycoviolobilin in P418 but not in P498. Subsequent treatment with dithiothreitol converts P552 into P418, whereas dithiothreitol reduces P498 to yield P420, a photoinactive form. Site-directed mutagenesis shows that the second Cys is essential for assembly of the photoactive holoprotein and that the photoactivity of this inert mutant is partially rescued by β-mercaptoethanol. These results suggest that the covalent attachment and detachment of a thiol, although not necessarily that of the second Cys, is critical for the reversible spectral blue shift and the complete photocycle. We propose a thiol-based photocycle, in which the thiol-modified P552 and P420 are intermediate-like forms.