Higher Detergent Concentrations Research Articles

Biocompatible and Safe Decellularized Spinach With Antibacterial and Wound Healing Activity.

Creating acellular vascularized constructs from animal and plant tissue is one of the well-known strategies for scaffold assembly. Decellularization takes an important position among these strategies. The most common method is chemical decellularization. This approach employs high concentrations of detergents, primarily Triton X-100, sodium dodecyl sulfate (SDS), and sodium hypochlorite (SH). In this work, novel techniques for decellularizing spinach were developed using detergents frequently utilized in laboratories. Spinach leaves were decellularized using Tween-20, SDS, and SH at low concentrations to generate an acellular plant matrix for tissue engineering. We measured the quantities of DNA and protein, as well as the decellularization using hematoxylin and eosin (H&E) staining. The biocompatibility and capacity of the biostructures to stimulate fibroblast wound healing were assessed using MTT and the Scratch assay. The antibacterial activity of the scaffolds was also tested against a gram-positive bacterium, Staphilococcus aureus, which is a common pathogen associated with wound healing. The best shape, evident vascularization, and good biocompatibility were seen in the Tween-20 decellularized samples at 1% concentration at 21°C and 37°C through the enhancement of cell proliferation and wound healing. In terms of antibacterial activity, all scaffold samples had a significant effect on Staphilococcus aureus, where the number of bacterial colonies in all six scaffold groups became zero after 4 h of treatment. The scaffolds also showed a 100% kill rate on Staphilococcus aureus, which could avoid wound infection during the repair process, and that can be suggested as a scaffold for tissue engineering applications and an important constituent for pharmacological activities.

Generational Diet-Induced Obesity Remodels the Omental Adipose Proteome in Female Mice.

Obesity, a complex condition that involves genetic, environmental, and behavioral factors, is a non-infectious pandemic that affects over 650 million adults worldwide with a rapidly growing prevalence. A major contributor is the consumption of high-fat diets, an increasingly common feature of modern diets. Maternal obesity results in an increased risk of offspring developing obesity and related health problems; however, the impact of maternal diet on the adipose tissue composition of offspring has not been evaluated. Here, we designed a generational diet-induced obesity study in female C57BL/6 mice that included maternal cohorts and their female offspring fed either a control diet (10% fat) or a high-fat diet (45% fat) and examined the visceral adipose proteome. Solubilizing proteins from adipose tissue is challenging due to the need for high concentrations of detergents; however, the use of a detergent-compatible sample preparation strategy based on suspension trapping (S-Trap) enabled label-free quantitative bottom-up analysis of the adipose proteome. We identified differentially expressed proteins related to lipid metabolism, inflammatory disease, immune response, and cancer, providing valuable molecular-level insight into how maternal obesity impacts the health of offspring. Data are available via ProteomeXchange with the identifier PXD042092.

Sustainable kitchen wastewater treatment with electricity generation using upflow biofilter-microbial fuel cell system.

The microbial fuel cell (MFC) is considered a modern technology used for treating wastewater and recovering electrical energy. In this study, a new dual technology combining MFC and a specialized biofilter was used. The anodic materials in the system were crushed graphite, either without coating (UFB-MFC) or coated with nanomaterials (nano-UFB-MFC). This biofilter served as a barrier to retain and remove turbidity and suspended solids, while also facilitating the role of bacteria in the removal of organic pollutants, phosphates, nitrates, sulfates, oil and greases. The results demonstrated that both systems exhibited high efficiency in treating kitchen wastewater, specifically greywater and dishwashing wastewater with high detergent concentrations. The removal efficiencies of COD, oil and grease, suspended solids, turbidity, nitrates, sulfates, and phosphates in first UFB-MFC were found to be 88, 95, 89, 86, 87, 75, and 94%, respectively, and in Nano-UFB-MFC were 86, 99, 95, 91, 81, 88, and 95%, respectively, with a high efficiency in recovering bioenergy reaching a value of 1.8 and 1.5Am-3, respectively. The results of this study demonstrate the potential for developing MFC and utilizing it as a domestic system to mitigate pollution risks before discharging wastewater into the sewer network.

An update on a technical method of cartilage decellularization: (a physical-based protocol)

Introduction: Despite advances in orthopedic surgery, the lack of effective conventional treatment for cartilage defects has led to research in cartilage tissue engineering. One of the interesting topics is the use of decellularized extracellular matrix (ECM) as a suitable natural scaffold that supports the growth and function of cells cultured in it. A concern with decellularization protocols, especially those with high detergent concentrations, is the disruption of native ECM, which has deleterious effects on subsequent scaffold recellularization. Therefore, this study focused on optimizing cartilage decellularization by physical methods without the use of ionic detergents. Methods: The bovine tracheal cartilage fragments were decellularized by a combination of 8 cycles of freeze-thaw and ultrasound techniques. Then, the tissues were immersed and shaken in 0.25% trypsin for 24 hours. Efficient cell removal and preservation of ECM were confirmed by histological and cytocompatibility assessments. The in-vivo studies were performed to evaluate the biocompatibility and bioactivity of the scaffold. Results: The histological assessments indicated the appropriate cytocompatibility and the fibroblast cell culture study demonstrated that cells were able to proliferate and migrate on the decellularized cartilage. In-vivo evaluation also showed a reduced adverse immune response, including leukocyte infiltration into the ECM. Conclusions: These results suggest that a cartilage scaffold created using a physical decellularization protocol that efficiently removes cells while preserving the native ECM can be a suitable scaffold for cartilage reconstruction. The main advantage of this protocol is the absence of potentially toxic chemicals in the tissues.

Evaluating the performance of surfactant and charcoal-based cleaning products to effectively remove PAHs from firefighter gear

Firefighters regularly respond to fire scenes where a mixture of chemicals including volatile, semi-volatile, and nonvolatile compounds are present in smoke and soot. Polycyclic aromatic hydrocarbons (PAHs) are common contaminants at fire scenes that may be deposited on the gear and the individual firefighter. Laundering is a common approach for the decontamination of contaminated gear. Surfactants are widely used by firefighters during laundering to remove PAHs as they are generally non-toxic and biodegradable. The removal of PAHs depends on the surfactant types, chemistries, and concentrations. This study evaluated the effect of surfactant concentrations to remove persistent contaminants like PAHs from turnout gear. The cleaning performance of different types of surfactants was also evaluated. Outer shell fabrics were contaminated with a standard mixture of 16 PAH compounds, and two commercial detergents were used at different concentrations. Additionally, the cleaning efficacy of eight commercially available regular and charcoal-based cleaning products was also determined against PAHs at a single surfactant concentration. For the decontamination method, a bench-scale washing procedure simulating the National Fire Protection Assocation 1851 laundering process was used. The removal efficacy of high molecular weight (HMW) PAHs were found to be lower compared to the low molecular weight PAHs for any type or any concentration of detergent. Our research also showed that the recommended surfactant concentrations provided by detergent manufacturers can be ineffective at removing the HMW PAHs from heavily contaminated fabric. With 1 mL of detergent in a 100-mL bath, which is multiple times higher than recommended amount, only 40% of HMW PAHs were removed. The cleaning efficacy can be increased to above 90% by using higher concentrations of detergents. This research shows that firefighters may need to use a higher concentration of detergent than the recommended amount to effectively remove PAHs from the gear. All the regular and charcoal-based detergents were able to remove PAHs effectively from contaminated fabrics when a higher concentration of detergent was used.

Effect of different decellularization protocols on reendothelialization with human cells for a perfused renal bioscaffold of the rat

BackgroundScaffolds for tissue engineering can be received by whole organ decellularization while maintaining the site-specific extracellular matrix and the vascular tree. One among other decellularization techniques is the perfusion-based method using specific agents e.g. SDS for the elimination of cellular components. While SDS can disrupt the composition of the extracellular matrix and impair the adherence and growth of site-specific cells there are indications that xenogeneic cell types may benefit from protein denaturation by using higher detergent concentrations. The aim of this work is to investigate the effect of two different SDS-concentrations (i.e. 0.66% and 3%) on the ability of human endothelial cells to adhere and proliferate in an acellular rat kidney scaffold.Material and methodsAcellular rat kidney scaffold was obtained by perfusion-based decellularization through the renal artery using a standardized protocol including SDS at concentrations of 0.66% or 3%. Subsequently cell seeding was performed with human immortalized endothelial cells EA.hy 926 via the renal artery. Recellularized kidneys were harvested after five days of pressure-controlled dynamic culture followed sectioning, histochemical and immunohistochemical staining as well as semiquantitative analysis.ResultsEfficacy of decellularization was verified by absence of cellular components as well as preservation of ultrastructure and adhesive proteins of the extracellular matrix. In semiquantitative analysis of recellularization, cell count after five days of dynamic culture more than doubled when using the gentle decellularization protocol with a concentration of SDS at 0.66% compared to 3%. Detectable cells maintained their endothelial phenotype and presented proliferative behavior while only a negligible fraction underwent apoptosis.ConclusionRecellularization of acellular kidney scaffold with endothelial cells EA.hy 926 seeded through the renal artery benefits from gentle decellularization procedure. Because of that, decellularization with a SDS concentration at 0.66% should be preferred in further studies and coculture experiments.

Genome-Based Analysis of Virulence Factors and Biofilm Formation in Novel P. aeruginosa Strains Isolated from Household Appliances.

In household washing machines, opportunistic pathogens such as Pseudomonas aeruginosa are present, which represent the household as a possible reservoir for clinical pathogens. Here, four novel P. aeruginosa strains, isolated from different sites of household appliances, were investigated regarding their biofilm formation. Only two isolates showed strong surface-adhered biofilm formation. In consequence of these phenotypic differences, we performed whole genome sequencing using Oxford Nanopore Technology together with Illumina MiSeq. Whole genome data were screened for the prevalence of 285 virulence- and biofilm-associated genes as well as for prophages. Linking biofilm phenotypes and parallelly appearing gene compositions, we assume a relevancy of the las quorum sensing system and the phage-encoded bacteriophage control infection gene bci, which was found on integrated phi297 DNA in all biofilm-forming isolates. Additionally, only the isolates revealing strong biofilm formation harbored the ϕCTX-like prophage Dobby, implicating a role of this prophage on biofilm formation. Investigations on clinically relevant pathogens within household appliances emphasize their adaptability to harsh environments, with high concentrations of detergents, providing greater insights into pathogenicity and underlying mechanisms. This in turn opens the possibility to map and characterize potentially relevant strains even before they appear as pathogens in society.

Optimization of detergents in solubilization and reconstitution of Aquaporin Z: A structural approach

BackgroundThe exceptional capacities of aquaporins in terms of water permeation and selectivity have made them an interesting system for membrane applications. Despite the multiple attempts for immobilizing the aquaporins over a porous substrate, there is a lack of studies related to the purification and reconstitution steps, principally associated with the use of detergents in solubilization and destabilization steps. This study analyzed the effect of detergents in Aquaporin Z solubilization, considering the purity and structural homogeneity of the protein. MethodsThe extraction process was optimized by the addition of detergent at the sonication step, which enabled the omission of the ultracentrifugation and resuspension steps. Two detergents, Triton X-100, and octyl-glucoside were also evaluated. Destabilization mediated by detergents was used as reconstitution method. Saturation and solubilization points were defined by detergent concentration and both, liposomes and proteoliposomes, were analyzed by size distribution and permeability assays. Detergent removal with Bio-beads was also analyzed. ResultsOctyl glucoside ensures structural stability and homogeneity of Aquaporin Z. However, high concentrations of detergents induce the presence of defects in proteoliposomes. While saturated liposomes create homogeneous and functional structures, solubilized liposomes get affected by a reassembly process, creating vesicle defects with anomalous permeability profiles. ConclusionsDetergent concentration affects the structural conformation of proteoliposomes in the reconstitution process. General significanceSince the destabilization process is dependent on vesicle, detergent, and buffer composition, optimization of this process should be mandatory for further studies. All these considerations will allow achieving the potential of Aquaporins and any other integral membrane protein in their applications for industrial purposes.

Pengolahan Air Sungai Menggunakan Slow Sand Filter Sistem Downflow dalam Menurunkan COD dan BOD

The raw water used as drinking water is currently contaminated with industrial, domestic, and agricultural waste, resulting in high concentrations of nitrogen, phosphate, detergent, COD and pesticides. Slow sand filters can be used as an effort to treat drinking water. This study aims to determine the effect of the variables used on the reactor performance for the removal of COD and BOD nutrients. In this study, the variation of gravel (with and without gravel) is used, so that the performance of each type of reactor can be seen. While the parameters to be measured are COD and BOD. The results showed that SSF gave a good reduction performance in reducing BOD up to 60% and COD up to 68.33% on the 6th day.

Purification of Crimean Congo Hemorrhagic Fever Virus (CCHFV) Nucleocapsid Protein Using Detergent Gradient and Free Thawing.

Protein aggregation remains a major challenge in the purification of recombinant proteins in both eukaryotic and prokaryotic expression systems. One such protein is the nucleocapsid protein of Crimean Congo Hemorrhagic fever virus (CCHFV), which has high aggregation tendency and rapidly precipitates upon purification by NiNTA chromatography. Using the detergent gradient purification approach reported here, the freshly purified protein by NiNTA chromatography was mixed with the dilution buffer containing a high detergent concentration, followed by overnight freezing at -80 °C. Thawing the resulting mixture at room temperature triggered the formation of a detergent concentration gradient containing the active protein in the low detergent concentration zone towards the top of the gradient. The inactive aggregates migrated to the high detergent concentration zone towards the bottom of the gradient. The method prevented further aggregation and retained the activity of the native protein despite numerous freeze-thaw cycles. This simple approach creates an appropriate microenvironment towards the top of the gradient for correctly folded proteins, and it prevents aggregation by rapidly separating the preformed early aggregates from the correctly folded protein in the mixture. This unique approach will be of potential importance for the biotechnological industry, as well as other fields of protein biochemistry that routinely purify recombinant proteins and face the challenges of protein aggregation. Graphical abstract.

A novel approach for the purification of aggregation prone proteins.

The protein aggregation is one of the major challenges of the biotechnological industry, especially in the areas of development and commercialization of successful protein-based drug products. The inherent high aggregation tendency of proteins during various manufacturing processes, storage, and administration has significant impact upon the product quality, safety and efficacy. We have developed an interesting protein purification approach that separates the functionally active protein from inactive aggregates using a detergent concentration gradient. The C-terminally His tagged nucleocapsid protein of Crimean Congo Hemorrhagic fever virus (CCHFV) has high aggregation tendency and rapidly precipitates upon purification by NiNTA chromatography. Using the new purification approach reported here, the freshly purified protein by NiNTA chromatography was further processed using a detergent gradient. In this new purification approach the active protein is retained in the low detergent concentration zone while the inactive aggregates are promptly removed by their rapid migration to the high detergent concentration zone. The method prevented further aggregation and retained the RNA binding activity in the native protein despite numerous freeze thaw cycles. This simple approach prevents protein aggregation by rapidly separating the preformed early aggregates and creating the appropriate microenvironment for correctly folded proteins to retain their biological activity. It will be of potential importance to the biotechnological industry and other fields of protein biochemistry that routinely face the challenges of protein aggregation.

Influence of the Linear Alkylbenzene Sulfonate (LAS) on hematological and biochemical parameters of Nile Tilapia, Oreochromis niloticus

The acute toxicity of household detergent (Ariel) on blood parameters and histology of Oreochromis niloticus was investigated using static bioassay for 96 h. Linear alkylbenzenesulfonate (LAS)is an anionic surfactant widely used in detergents and cleaners, both in industrial and household applications.LAS contaminating aquatic ecosystems as a potential toxic pollutant, was investigated in the present study for acute toxicity.The fish samples were divided into six groups, including 20 fish in each group. Normal feed was given to control group without detergents treatment. Hematological parameters (RBC count, Hb, Ht and platelets) were significantly declined, while WBC count showed a highly significant increase. Compared with the control group, significant elevation of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was recorded in fish treated with different concentrations of detergent. Catalase (CAT), Superoxide dismutase (SOD) activities and Reduced Glutathione (GSH) concentration showed a highly significant reduction. Total proteins showed significant decrease, while total lipids, cholesterol and triglycerides significantly increased. The mean lethal concentration (LC50) for 96 h of Ariel was at concentration 10 mg/L. Relative percentage of detergent residues in fish muscles was increased with higher detergent concentrations. In conclusion, exposure to detergents resulted in great alterations in the histological structure of liver and gills.

USING IRON-CONTAINING METAL OXIDE AS CATALYST FOR HETEROGENEOUS FENTON PROCESS IN TEXTILE INDUSTRY WASTEWATER

The unconscious use of surface and ground waters and the rapid pollution of water, which is the main source of life for all living creatures as a result of drought due to global warming, pose a serious problem. The rapidly increasing world population and the need for clean water have brought up a global water crisis. The textile industry is one of the largest producers of wastewater in the world. Textile industry wastewater contains high amounts of non-biodegradable organic compounds, high concentrations of dyestuffs, salt, detergent and soap. Therefore, it is of great importance to remove organic pollutants in this wastewater. Since traditional methods are insufficient to remove organic compounds in wastewater, advanced treatment methods are required. Advanced oxidation processes (AOPs) are one of the alternative treatment methods preferred in recent years. In this study, color removal from textile industry wastewater was researched by the heterogeneous Fenton process, which is an advanced oxidation process. The parameters such as catalyst dosage, pH, hydrogen peroxide concentration, temperature, reaction time and mixing speed that effect heterogeneous Fenton processes were investigated. Under optimum experimental conditions, the color removal efficiency was achieved as 87%.

Stabilization and improved properties of Salipaludibacillus agaradhaerens alkaline protease by immobilization onto double mesoporous core-shell nanospheres

In this study, serine alkaline protease from halotolerant alkaliphilic Salipaludibacillus agaradhaerens strain AK-R was purified and immobilized onto double mesoporous core-shell silica (DMCSS) nanospheres. Covalent immobilization of AK-R protease onto activated DMCSS-NH2 nanospheres was more efficient than physical adsorption and was applied in further studies. DMCSS-NH2 nanospheres showed high loading capacity of 103.8 μg protein/mg nanospheres. Relative to free AK-R protease, the immobilized enzyme exhibited shifts in the optimal temperature and pH from 60 to 65 °C and pH 10.0 to 10.5, respectively. While the soluble enzyme retained 47.2% and 9.1% of its activity after treatment for 1 h at 50 and 60 °C, the immobilized protease maintained 87.7% and 48.3%, respectively. After treatment for 2 h at pH 5 and 13, the immobilized protease maintained 73.6% and 53.4% of its activity, whereas the soluble enzyme retained 32.9% and 1.4%, respectively. Furthermore, the immobilized AK-R protease showed significant improvement of enzyme stability in high concentration of NaCl, organic solvents, surfactants, and commercial detergents. In addition, the immobilized protease exhibited a very good operational stability, retaining 79.8% of its activity after ten cycles. The results clearly suggest that the developed immobilized protease system is a promising nanobiocatalyst for various protease applications.

Iron-crosslinked Rososome with robust stability and high drug loading for synergistic cancer therapy

Development of liposomal nanomedicine with robust stability, high drug loading and synergistic efficacy is a promising strategy for effective cancer therapy. Here, we present an iron-crosslinked rosmarinic liposome (Rososome) which can load high contents of drugs (including 25.8% rosmarinic acid and 9.04% doxorubicin), keep stable in a high concentration of anionic detergent and exhibit synergistic anti-cancer efficacy. The Rososomes were constructed by rosmarinic acid-lipid conjugates which not only work synergistically with doxorubicin by producing reactive oxygen species but also provide catechol moieties for the iron cross-linkages. The cross-linkages can lock the payloads tightly, endowing the crosslinked Rososome with better stability and pharmacokinetics than its non-crosslinked counterpart. On the syngeneic mouse model of breast cancer, the iron-crosslinked Rososomes exhibit better anticancer efficacy than free rosmarinic acid, doxorubicin, non-crosslinked Rososome and commercial liposomal formulation of doxorubicin (DOXIL). This study introduces a novel strategy for the development of liposomes with robust stability, high drug loading and synergistic anti-cancer efficacy.

Hematological response of Tilapia (Oreochromis niloticus) in Laundry wastewater

The high concentration of detergent in the aquatic ecosystem potentially affects the physiological condition of the fish by disrupting the respiration process and changing the concentration of blood components and chemistry. The purpose of this study was to determine the condition of the hematological parameters of tilapia (Oreochromis niloticus) exposed to wastewater from the laundry industry. Each treatment was stocked with five fish per aquarium (50 x 30 x 30 cm). This study used a completely randomized design (CRD) technique with six treatments and three replications: P0 (0%) as a control, P1 (1%), P2 (2%), P3 (3%), P4 (4%) and P5 (5%) with each treatment exposed to a specific concentration of wastewater and residues. The results showed that the hemoglobin levels of treatments were decreasing, with the lowest mean of hemoglobin level found in the P2 (7.05 gr%), and the lowest concentration on the 30th day was 7.71 gr%. There were no significant effects of wastewater to the number of erythrocytes and leucocytes among treatments (P>0.05). While there were increasing levels of hematocrit, the largest mean level was found in the P4 treatment with a value of 24.11%, and the largest mean on the 20th day of observation showed a value of 23.51%. Wastewater from the laundry industry can affect the hematological condition of tilapia by decreasing the hemoglobin concentration and increasing the hematocrit levels above the normal condition.

FiTAc-seq: fixed-tissue ChIP-seq for H3K27ac profiling and super-enhancer analysis of FFPE tissues

Fixed-tissue ChIP-seq for H3K27 acetylation (H3K27ac) profiling (FiTAc-seq) is an epigenetic method for profiling active enhancers and promoters in formalin-fixed, paraffin-embedded (FFPE) tissues. We previously developed a modified ChIP-seq protocol (FiT-seq) for chromatin profiling in FFPE. FiT-seq produces high-quality chromatin profiles particularly for methylated histone marks but is not optimized for H3K27ac profiling. FiTAc-seq is a modified protocol that replaces the proteinase K digestion applied in FiT-seq with extended heating at 65 °C in a higher concentration of detergent and a minimized sonication step, to produce robust genome-wide H3K27ac maps from clinical samples. FiTAc-seq generates high-quality enhancer landscapes and super-enhancer (SE) annotation in numerous archived FFPE samples from distinct tumor types. This approach will be of great interest for both basic and clinical researchers. The entire protocol from FFPE blocks to sequence-ready library can be accomplished within 4 d.

A study of the influence of temperature and detergent concentration on the removal of lipstick stains using air‐supersaturated water, where fine bubbles are generated by ultrasonic stimulation

AbstractThe formation of fine bubbles with a diameter between 10 and 20 μm was observed upon the passage of air‐supersaturated water, in which air is pressurized and dissolved, through a water passage in which supersonic stimulation was given to the water. It was confirmed that the inside diameter of the fine bubbles increases as the distance from the ultrasonic stimulation point becomes greater. In verifying the efficiency of cleaning a lipstick stain, it was shown that by using water containing fine bubbles, there is a possibility of obtaining a cleaning efficiency almost equivalent to that achieved by cleaning with water of a high temperature or with a high concentration of detergent, even at a low temperature, with low detergent concentration. It was shown that the efficiency of cleaning the oil components, which were made easily soluble in water, had become extremely high, when using water containing fine bubbles. In conditions where the melting of the oil component itself is not accelerated (that is, at a low temperature, without using detergents), the cleaning efficiency of water containing fine bubbles tended to be increased compared with that of water not containing fine bubbles.

Effect of detergents on alkaline invertase and alkaline phosphatase activity of fungi Mucor plumbeus Bonord, 1864, Aspergillus Niger Tiegh, 1867 and Trichoderma harzianum Rifai, 1969

Due to their diverse metabolic potential, many filamentous fungi have a great ability for degradation of different waste substances. The present research was aimed to investigate ability of fungi Mucor plumbeus, Aspergillus Niger and Trichoderma harzianum, isolated from sewer and industrial waste water, to conduct degradation of high concentrations of commercial detergent (0.3%). Within enzyme extracts of samples taken during period of 3-16 days, activity of alkaline invertase and alkaline phosphatase was followed spectrophotometrically at room temperature. Obtained results proved that all examined fungi affected degradation of detergent. Quality (inhibition/stimulation) and quantity of action of detergent on activity of investigated enzymes depended on fungal species and incubation period. The highest inhibiting effect of detergent was recorded on 9th day of incubation in samples of T. harzianum and A. Niger, while its most obvious stimulating effect was noticed on 3rd day of incubation in samples of T. harzianum and M. plumbeus. Investigated fungi can be used for purification of waste water containing high concentrations of detergent.

Spectroscopic Studies on the Conformational Stability of Hemocyanin of Pila virens (Lamarck, 1822) in the Presence of Temperature and Detergents

Gastropods are endowed with a soluble copper containing, blue coloured respiratory protein known as hemocyanin. In the present study, for the first time, hemocyanin from Pila virens, a fresh water gastropod endemic to Indian sub-continent was studied for its conformational stability in the presence of temperature and various detergents by different spectroscopic techniques viz. UV–Vis, fluorescence and circular dichroism spectroscopy. Results from UV–Vis spectroscopy revealed that, absorbance corresponding to active site moieties were decreased with increase in temperature from 30 to 70 °C. Whereas ‘thermal reversibility’ studies showed the increase in absorbance of active site with decrease in temperature from 70 to 20 °C (is an indicative of the characteristic feature of hemocyanins—that oxygen is reversibly bound to the copper in the active site). Results from CD spectroscopy revealed that the temperature-induced conformational changes on the secondary structure of hemocyanin was reversible up to 70 °C. Beyond 80 °C, the protein was completely denatured and assumed an increased random coil conformation. Hemocyanin in the presence of high concentration of detergents lost its structure as evident from UV–Vis fluorescence (evident from the red shifts in the emission maxima accompanied by quenching in emission intensities) and CD spectroscopy. Stability of active site moieties in the presence of different detergents was also studied by UV–Vis spectroscopy. The biophysical techniques gave insights into the varied effects of each of the different denaturants on the conformational stability of hemocyanin from Pila virens.