Effect Of Incubation Period Research Articles

Visible and near infrared spectroscopy for predicting soil nitrogen mineralization rate: Effect of incubation period and ancillary soil properties

Soil nitrogen mineralization rate (SNMR) influences crop N uptake and nitrate leaching leading to environmental pollution. This study aims at (i) examining whether visible and near-infrared reflectance spectroscopy (vis-NIRS) can predict SNMR and (ii) investigating if incubation periods and ancillary soil attributes can improve the prediction accuracy. Total 133 soil samples collected from seven fields were incubated under aerobic conditions for 60 days with seven batches of sub-samples. Mineral N was measured at regular time intervals and soil samples were scanned using a vis-NIRS sensor (Tec5 Technology, Germany) parallelly. SNMR was determined by fitting a zero-order kinetic to the net mineralized N as a function of the incubation time. Soil total nitrogen (TN), total carbon (TC) and electrical conductivity (EC) were determined once. Partial least squares regression (PLSR) models were calibrated individually for each field both for vis-NIR spectra and its combinations with TN, TC and EC. Six out of seven batches of sub-samples were used for calibrating PLSR when remaining one batch was used for model validation, and it rotated across all seven batches. Vis-NIRS alone predicted SNMR with moderate accuracy in five of seven fields (coefficient of determination, 0.53 ≤ R2 ≥ 0.66, ratio of prediction to deviation, 1.51 ≤ RPD ≥ 1.76), while models were poor in two fields (R2 = 0.23–0.26, RPD = 1.18 – 1.20). Inclusion of soil TC, TN and/or EC was expected to improve accuracy, but improvements varied across fields (R2 = 0.23–0.79, RPD = 1.18 – 2.26). Similarly, the incubation period increased vis-NIRS prediction accuracy, but frequently occurred among 2nd to 6th batches (R2 = 0.35–0.82, RPD = 1.28 – 2.44). Even incorporating secondary properties and increasing incubation duration hardly improved predictions, improvement can be compromised since it is not significant mostly and often underperformed or remained unchanged. Considering the time and effort required to incubate and analyze soil properties, this study suggests using a vis-NIRS sensor to estimate SNMR in fresh soil conditions i.e., without incubation and incorporation of secondary properties.

Isolation and optimisation of polyphosphate accumulating bacteria for bio-treatment of phosphate from industrial wastewater

ABSTRACT Phosphorus in wastewater influents is a global issue. Controlling eutrophic water is crucial. Biological phosphorus removal is an economically and environmentally sustainable method for removing phosphorus from wastewater. This study aims to isolate and improve the capacity of aerobic phosphorus-removing bacteria to reduce excessive phosphate concentrations in the environment. Only three out of fourteen bacterial isolates demonstrated the highest phosphate removal efficiency using Toluidine blue-O. Klebsiella pneumoniae 6A, Klebsiella quasipneumoniae 6R, and Enterobacter mori 8R were isolated from activated sludge and identified by 16srRNA. In a single-factor experiment, the effect of incubation periods, phosphate concentrations, carbon sources, sodium acetate concentrations, temperature, pH, and irradiation dosages were studied. Seventy-two hours of incubation, 55 mg/L PO4, sodium acetate as the carbon source, 30°C and pH 7 resulted in maximum phosphorus removal. After optimising the parameters, the removal efficiency of Klebsiella pneumoniae 6A, Klebsiella quasipneumoniae 6R, and Enterobacter mori 8R increased from 73.5% to 85.1%, 79.1% to 98.1%, and 80.6% to 91.9%, respectively. Gamma irradiation showed significant results only in Klebsiella pneumoniae 6A where 100 Gy increased the phosphorous removal efficiency from 85.1% to 100%. Immobilised mixed culture of the three strains adapted better to 100 mg/L Phosphorus than pure cells. Therefore, this technique holds great new promise for phosphorus-contaminated sites bioremediation.

Screening of Chitinolytic Microfungi and Optimization of Parameters for Hyperproduction of Chitinase Through Solid-State Fermentation Technique.

This study is intended for the production of chitinase enzyme from locally isolated fungal strains. Out of 10 isolated fungal strains from district Gujrat, Punjab, Pakistan, Aspergillus terreus SB3 (accession number ON738571) was found with maximum chitinolytic potential (80.8 U/mL/min). By applying central composite design (CCD) through response surface methodology (RSM) under solid-state fermentation (SSF), eight nutritional and physical parameters were optimized. Among these, temperature, substrate concentration, and pH were found as significant factors toward chitinase production in the first phase. Moisture and nitrogen source were found as significant factors during second phase of chitinase production. The effect of incubation period, inoculum size, and magnesium source was observed as non-significant. The chitinase activity was successfully enhanced more than 2 folds up to 198.5 U/mL/min at optimized conditions of 35°C temperature, 4.5 pH, 20g substrate concentration, 4-day incubation period, 55% moisture content, 4.5mL inoculum size, 0.25g ammonium sulfate, and 0.30g magnesium sulfate using RSM design. It was also found that Ganoderma lucidum (bracket fungus) has more potential to be used for the production of chitinase compared to fish scales. The present study exhibited Aspergillus terreus SB3 (ON738571) as a potential indigenous strain capable for hyperproduction of chitinase through cheap fermentation technology that might be employed for the eradication of chitin-based sea waste to remove the marine pollution.

The Effect of Incubation Period on the Shelf Life of Kefir Beverage Prepared with Kefir Culture

The aim of this study was to examine the effect of different incubation times on the shelf life of kefir. For this purpose, kefir samples were first incubated for 8, 12, 18, 24 and 36 hours, creating 5 different kefir groups. It was then stored at 4±1°C for 21 days.. Microbiological (Lactobacillus spp., Lactococcus spp., and yeast), chemical (pH, titratable acidity), and sensory analyses of the kefir groups were performed during the storage period. The Lactobacillus (7.80 log10 cfu/mL), Lactococcus (7.30 log10 cfu/mL) and yeast (6.14 log10 cfu/mL) counts were higher in the kefir groups that were incubated for a long time, and changes were observed during the storage process depending on the incubation time. It was determined that the titratable acidity became higher as the length of the incubation and storage time increased. The results showed that the incubation time had an effect on Lactobacillus spp. and Lactococcus spp. counts, pH and acidity (P

Husk-like Zinc Oxide Nanoparticles Induce Apoptosis through ROS Generation in Epidermoid Carcinoma Cells: Effect of Incubation Period on Sol-Gel Synthesis and Anti-Cancerous Properties

This study effectively reports the influence of experimental incubation period on the sol-gel production of husk-like zinc oxide nanoparticles (ZNPs) and their anti-cancerous abilities. The surface morphology of ZNPs was studied with the help of SEM. With the use of TEM, the diameter range of the ZNPs was estimated to be ~86 and ~231 nm for ZNPA and ZNPB, prepared by incubating zinc oxide for 2 and 10 weeks, respectively. The X-ray diffraction (XRD) investigation showed that ZNPs had a pure wurtzite crystal structure. On prolonging the experimental incubation, a relative drop in aspect ratio was observed, displaying a distinct blue-shift in the UV-visible spectrum. Furthermore, RBC lysis assay results concluded that ZNPA and ZNPB both demonstrated innoxious nature. As indicated by MTT assay, reactive oxygen species (ROS) release, and chromatin condensation investigations against the human epidermoid carcinoma (HEC) A431 cells, ZNPB demonstrated viable relevance to chemotherapy. Compared to ZNPB, ZNPA had a slightly lower IC50 against A431 cells due to its small size. This study conclusively describes a simple, affordable method to produce ZNP nano-formulations that display significant cytotoxicity against the skin cancer cell line A431, suggesting that ZNPs may be useful in the treatment of cancer.

Effect of incubation period on the glycosylated protein content in germinated and ungerminated seeds of mung bean (Vigna radiata (L.) Wilczek)

The effects of different incubation periods on the contents of amino acids, proteins, glycosylated proteins and metabolites in germinated and ungerminated mung bean seeds were investigated in this study. The study employs soaking of mung bean seeds in water under laboratory conditions at 28 °C for 3, 6, and 9 h, followed by germination for 12, 24, 36, and 48 h. Seeds collected from different period of imbibition and germination were subjected to total protein extraction for phytochemical analysis. Germination of the seeds was found to be most successful after 6 h of soaking (rather than 9 h of incubation). Hence, seeds imbibed for 6 h were further investigated for germination at 28 °C for 12, 24, 36, and 48 h. Total protein was extracted from both imbibed and germinated seeds, followed by trypsin digestion. Liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based peptide mass fingerprinting revealed 38 proteins in 6 h water-imbibed seeds and 50 proteins in 24 h germinated seeds. Among these, 16 were identified as glycosylated proteins and the maximum number of glycosylated proteins were detected in 6 h water-imbibed seeds and 24 h germinated seeds. Moreover, High Performance Liquid Chromatography (HPLC) was used to quantify amino acids from the extracted proteins. A total of 15 amino acids were detected, of which eight were essential and the remaining were non-essential; amino acid concentrations increased following 3, 6, and 9 h of imbibition when compared to the control. It was concluded from the study that seeds with 6 h of imbibition and 24 h of germination can be used as potential nutritional source of different amino acids, proteins, glycosylated proteins, and other bioactive metabolites in human diet.

Molecular Screening and Production of α-amylase from Fungal sp.

α-amylase producing Aspergillus sp. was isolated from decaying bread collected from bakery site located within Enugu metropolis, Enugu state. Standard microbiology, biochemical and molecular techniques (18s DNA sequencing) were used for confirmation of the fungal strain as Aspergillus tamari.. p-NPG infused nutrient broth confirmed Aspergillus tamarrii as prolific producer of α-amylase with rapid yellow colouration after forty eight hours of incubation. Solid state fermentation on rice bran matrix (SSF) system was used for the enzyme production. Cork borer of 2mm diameter was used throughout the production and optimization studies. Carbon sources including: Starch, wheatbran and sugarcane baggase were optimized, starch was found suitable for the protein production with highest α-amylase activity (91.71 μmol/min). Among the nitrogen sources optimized, peptone was found optimal for α-amylase production with activity of 90.34 μmol/min. pH 6.0 was found the best for the enzyme production. Effect of incubation period on the enzyme production showed the 4th day of fermentation as the peak day for α-amylase production from Aspergillus tamari. The results from this study have shown that Aspergillus tamari among other fungal isolates from mould bread collected from bakery sites in Enugu metropolis has the potential for α-amylase production in a commercial scale for both industrial and clinical applications..

An experimental approach for measuring carbon dioxide diffusion coefficient in water and oil under supercritical conditions

Several direct or indirect approaches have been proposed to measure diffusion coefficient of gases into liquids. The main complexity of indirect techniques such as pressure decay method is interpreting early pressure–time data which strongly affected by incubation period effect or convective instability. In the current approach, accurate apparatus and precise experimental setup including a high pressure and temperature PVT cell, a high precision Sanchez pump, heating and recording sub-system are implemented and a novel data analysis procedure is applied to modify pressure decay method. The effect of incubation period is reduced remarkably and diffusion coefficient of carbon dioxide in water in wide range of pressures and temperatures is determined and the effects of temperature, pressure and carbon dioxide phase alteration from gas to supercritical are investigated and the value of uncertainty is estimated. Furthermore, diffusion coefficient of CO 2 and methane in an oil sample from one of the Iranian southwest oil formations is determined precisely using the experimental approach while no incubation period is detected. The results showed that incubation period duration decreases with increasing diffusion coefficient. Additionally, when CO 2 state is gas, rate of increasing diffusion coefficient with pressure is decreased with temperature and when CO 2 state is supercritical, the rate of increasing diffusion coefficient with pressure is decreased significantly.

Response surface methodology: An effective optimization strategy for enhanced production of nitrile hydratase (NHase) by Rhodococcus rhodochrous (RS-6)

Nitrile hydratase is an enzyme which catalyze the hydration of nitriles into amide and their role as catalysts for acrylamide production in industries are well known. The present study aims at statistically optimizing physiological and nutritional parameters for NHase production from Rhodococcus rhodochrous (RS-6). The effect of incubation period, temperature, pH, carbon and nitrogen sources on the production of NHase was investigated by one factor at a time strategy. Further optimization process was carried out by response surface methodology for studying the interactive effect of these variables using central composite design. The optimized levels of variables obtained by statistical analysis were: incubation period 48 h, temperature 33 °C, pH 7.0, glycerol 1% and urea 0.75%, which resulted in maximum NHase production. The results of ANOVA were significant with the F-value of the model being 296.78, value of R2 is 0.9983 and the lack of fit test was not significant. The contour and response surface plots showed significant interaction between the variables. The NHase yield was enhanced up to 6.22 fold by statistical optimization using RSM. Thus, the developed experimental design is effective towards process optimization for NHase production from R. rhodochrous (RS-6).

Effects of Incubation Period and Fasting Time on the Duodenum Morphology from One-Day-Old Commercial Broiler Chicks: A Histomorphometrical Study

Effects of Incubation Period and Fasting Time on the Duodenum Morphology from One-Day-Old Commercial Broiler Chicks: A Histomorphometrical Study

Green ethanol production from cotton stalk

This study was initiated to produce green ethanol from cotton stalk lignocelluloses using separate hydrolysis and fermentation (SHF) process. The influence of sodium hydroxide (2%) pretreatment at different residence times was also investigated. The effect of incubation period, enzyme concentration, pH and substrate concentration during enzymatic saccharification on sugar yield and ultimately on the ethanol production was studied. The results show that pretreatment with 2% NaOH for 90 min at 121°C was most effective and gave the highest percentage of cellulose (78.2±1.64%) along with highest delignification rate (63.9%). During the enzymatic saccharification, under the optimized conditions, maximum concentration of reducing sugars of the hydrolyzate was 67.83 ± 0.35 g/L with the maximum saccharification efficiency of 78.06%. The total sugars were 77.48 ± 2.37 g/L and total phenols were found to be 1.6 mm. Fermentation was carried out by using the enzymatically saccharified hydrolyzate and monoculture of Saccharomyces cerevisiae. Highest ethanol concentration of 22.93 ± 1.74 g/L with 0.36 g/g ethanol yield was achieved after 48 h of incubation at 30°C, pH 5 and 3% inoculum size.

Effect of Incubation Periods, Medium Volumes and Explants Density on in Vitro Shoot Formation and Growth and Cost of Multiplication of Moris Pineapple (Ananas comosus L.)

This study was conducted to investigate the effect of combinations of four incubation periods (30, 45, 60 and 75 days), three explants density (one, two and three) and four volumes of medium (3, 6, 9 and 12 ml) in the in vitro shoot formation per explant, total shoots and total cost per liter of medium and cost per shoot of Moris pineapple. The highest shoot per explant was obtained in combination of three explants in 12 ml of medium incubated for 60 days, three explants in 9 and in 12 ml of medium and one explant in 3 and in 9 ml of medium incubated for 75 days but at different total shoots (2750, 3667, 2750, 3667 and 1222 shoots), total cost ( USA $25.85, 36.09, 28.39, 95.32 and 36.06) per liter of medium. Combinations in which three explants were used per culture resulted in lowest cost per shoot (1.0 cent) while combination in which one explant was used per culture, the cost per shoot was two to three times higher (2.6 and 2.9 cents). Combination of three explants, 3 ml of medium and 75 days of incubation, on the other hand, resulted in formation of only 6 shoots per explant (almost half of the highest obtained rate) but in highest total shoot per liter of medium (6000 shoots ) at highest total cost (USA $95.32) and intermediate cost per shoot (1.6 cents). Combination of three explants, 6 ml of medium and 75 days resulted in formation of 10 shoots per explant, production of 5000 shoots per liter of medium at lowest cost per shoot (1.0 cent) and intermediate total cost per liter of medium (USA $ 50.68). Using of one explant in 12 ml for 30 days resulted in formation of 5 shoots per explant, highest cost per shoot (5.0 cents) and lowest total shoots (417 shoots) and lowest total cost (USA $ 20.73) per liter of medium.

In Vitro Effect of Degradation in Sacco in the Rumen on the Anthelmintic Properties of Parkia Biglobosa and Pterocarpus Erinaceus on Haemonchus Contortus

Parkia biglobosa and Pterocarpus erinaceus are traditionally used to treat various ailments including helminth infections. This study was undertaken to evaluate the effect of the rumen degradability on anthelmintic properties of the two plants and to examine the possible role of tannins and/or polyphenols on these properties. Hydro-acetone extracts of in sacco degradability residues of P. biglobosa pods and P. erinaceus leaves were screened in vitro to determine the possible anti-parasitic effects against eggs and infective larvae of Haemonchus contortus. In addition, the possible involvement of tannins and/or polyphenols was examined by comparing the levels of inhibition of larval migration obtained with the same extracts, after of not addition of PVPP. Extracts of both plants induced significant egg hatch inhibition (p < 0.001). The effects were dose and incubation period dependent. Extracts of both plants have shown again remarkable larval migration inhibition compared to the PBS (p < 0.05). The effect of incubation period was not significant (p > 0.05). These results suggest that the plants did not lose their anthelmintic properties after rumen degradation. The use of the PVPP indicated for almost all of the extracts that tannins and/or polyphenols are largely involved in the effect. Complementary investigations are necessary to understand the metabolism of these plants in the digestive tract of the animals.

Effects of incubation period and Christmas Island rock phosphate with different rate of rice straw compost on phosphorus availability in acid soil

Phosphorus (P) availability is limited in tropical acid soil due to fixation of soluble inorganic P by aluminium (Al) and iron (Fe). Liming is one of the common ways to overcome this problem. However, this practice is not economical. The aim of this study was to evaluate the effects of laboratory incubation period and different rates of rice straw compost on the soil P availability. During the incubation study, Christmas Island rock phosphate (CIRP) fertilizer was amended with different rates of rice straw compost (5, 10, 15, and 20 t ha−1). Treatments were incubated in the laboratory for 30, 60, and 90 days. Application of rice straw compost with CIRP significantly increased soil available P at 30, 60, and 90 days of incubation, respectively. This implies rice straw compost plays an important role on soil P availability by decreasing the P adsorption due to the competing adsorption sites by organic anion and dissolving the mineral associated P by low-molecular-weight organic acids. Besides, rice straw compost also increased the soil pH, and, at the same time, reduced exchangeable acidity, exchangeable Al and Fe. As the soil pH increased, the compost effectively fixed the Al and Fe in the soil instead of P, thus increasing the P availability in the soil. However, there was no significant increase/loss of available P when the incubation time increases under treatments with rice straw compost. This implies the effectiveness of rice straw compost in minimizing the loss of P due to P fixation in soil and slow microbially mediated mineralization of soil organic P to inorganic P as incubation time increases. The findings suggest that the application of rice straw compost altered soil chemical properties in a way that enhanced the availability of P in the Rengam acidic soil.

Influence of Incubation Periods on Extractable Fluoride and Phosphorous at Different Exchangeable Sodium Percentage Levels

The laboratory incubation experiment was accomplished to study the effect of incubation periods on the extractable Fluoride (F) and Phosphorous (P) in soil at different exchangeable sodium percentage (ESP) levels. The soils of different ESP levels (30, 45 and 60) were prepared by applying different amount of sodium bicarbonate dissolved in distilled water to the calculated volume of saturation percentage of the soil. The observed ESP of the prepared soil was 27.1, 43.7 and 54.9. Incubation studies were conducted with fluoride levels ranging from 40 to 160 mg/kg and phosphorus levels from 12.5 to 50 mg/kg with incubation periods of 1, 3,7, 14, 21, 28 and 35 days at temperature of 25±2℃. Moisture was maintained at field capacity by addition of distilled water. The results showed that the extractability of fluoride in soil increased with increasing levels of F and P application along with increased ESP. The increase was 100 percent at ESP 54.9 compared to ESP 6.2 at F level of 160 mg/kg at 1st day of incubation period. Similarly, Olsen’s extractable phosphorus in soil increased with increasing levels of F, P and ESP. It increased by 66 percent at ESP 54.9 compared to ESP 6.2 at P level of 50 mg/kg at 1st day of incubation period. However, extractability of both F and P decreased with increase in incubation periods. Fluoride extractability decreased by 65 percent at ESP 6.2 and 25 percent at ESP 54.9 with increasing days from 1st to 35th day of incubation period. Olsen’s extractable phosphorus decreased by 37 percent at ESP 6.2 and 48 percent at ESP with increasing days from 1st to 35th day of incubation period.

Effect of incubation period on measurement of alkaline phosphatase enzyme activity at 37°C

Background: Laboratory analysis of alkaline phosphatase (ALP) from blood can detect abnormalities which reflect changes in certain health conditions, including liver disease and bone disease. Despite the importance of measuring different isoenzymes activity in different diseases, total ALP test is often requested, which is not specific to identify the type and the extent of tissue damage. ALP activity measurement at different incubation period may affect the actual measurement as ALP is a heat labile enzyme and specific ALP isoenzymes have different sensitivity to heat inactivation which creates lab to lab variability and significant change if particular isoenzyme is predominant in patient’s sera. Objectives: The study was performed to measure the loss of activity of ALP at different 37°C incubation periods during ALP measurement in serum. Materials and Methods: Quality control materials were analyzed for ALP enzyme activity in 2-amino, 2-methyl, 1-propanol buffer, and 4-nitrophenyl phosphate substrate with different incubation periods. Result: There is a gradual decrease in enzyme activity on increase of incubation period. Conclusion: The effect of incubation period on ALP activity measurement at 37°C may be clinically relevant if one of the isoenzymes is predominant in the patient sera.

Environmental parameters affecting biosorption of heavy metals by mixed culture of Aspergillus niger and Aspergillus flavus in industrial wastewater

This work was conducted to study the effect of some environmental conditions on bioremoval of heavy metals from industrial wastewater by consortium cultures of Aspergillus niger and A. flavus. Many procedures on the effect of incubation period, temperature, pH and concentration of metals were carried out in the laboratory. Results proved that the high culture growth and maximum heavy metals removal were after 21 days incubation at 30 oC, pH of 5.4 and metal concentration at 10 ppm. While the lowest culture growth and minimum heavy metals removal were after 7 days incubation at 5 oC , pH of 10.8 and metal concentration at 100 ppm.

Biodegradation of Atrazine by Bacteria Isolated from Lotic Water

Background: The persistence of toxic herbicides in water and soil has been considered to be potential environmental concern. As atrazine is still used in Nigeria as a major herbicide, a continuous search for atrazine degrading microorganisms is required. Objectives: The effects of incubation period on growth and atrazine degradation by bacteria isolated from a lotic water in the Niger Delta were evaluated and also determined were the effects of temperature and anaerobic incubation on atrazine removal by the most efficient isolate. Methods: The bacteria capable of degrading atrazine were isolated from the lotic water using enrichment technique. The optimal incubation period for growth and atrazine degradation by the isolates was assessed by growing the isolates in Mineral salts medium containing 100 mg/L atrazine for 30 days at 35oC. The flasks inoculated with the most efficient isolate were also incubated anaerobically and at varying temperatures (25, 30, 35 and 40oC). Cultures were withdrawn every 5 days and growth and atrazine concentration measurements were carried out using standard plate count and HPLC respectively. Results: Atrazine degrading bacteria of the genera Pseudomonas, Bacillus and Micrococcus were isolated with Pseudomonas sp. as the most efficient isolate. Incubation time of 20 days was Original Research Article Ariole and Abubakar; JALSI, 2(3): 119-125, 2015; Article no.JALSI.2015.013 120 observed as optimum for growth of the three isolates. Pseudomonas sp. gave the highest atrazine degradation rate of 82.67% followed by Bacillus sp. (75.33%) and Micrococcus sp. (69.33%) at the end of 30 days. Atrazine was degraded at reduced rate under anaerobic condition and temperature of 35oC was optimum for atrazine degradation by Pseudomonas sp. Conclusion: These atrazine degrading strains may be useful in bioremediation of contaminated wastewater.

In vitro Antagonistic Activity of Candida albicans against Filamentous Fungi

Background: Candida albicans is a special type of yeast that is naturally found on the surface of various mucosal layers of the human body. Biofilms forming is well known synergistic relationship between C. albicans and various organisms with no or little aggressive acted toward each other. Methods and findings: C. albicans that isolated from patient with cutaneous candidiasis was tested for antifungal activity against six species of filamentous fungi by measuring of percentage inhibition. The effects of incubation periods and different concentrations of glucose on the antifungal activity of C. albicans were also tested. The percentage inhibition of growing filamentous fungi had been observed at high values when they cultivated on media that previously cultured with C. albicans than when both of fungi and C. albicans cultured at the same time, especially at low concentrations of glucose. Mucor spp. and C. sitophila had been affected by C. albicans only when they cultivated on media that previously cultured with C. albicans. Conclusion: C. albicans had been shown the ability to inhibit various species of filamentous fungi. Incubation periods and glucose concentrations had been effected on the inhibitory action of C. albicans against other fungi.

Fine characterization of glucosylated human IgG by biochemical and biophysical methods

Nonenzymatic glycosylation of proteins finally generates advanced glycation end products (AGEs). The Schiff's base and Amadori adduct are stages of early glycation. AGE-modified IgG may undergo conformational alterations and the final entity of the process may be involved in the pathogenesis of Rheumatoid Arthritis (RA). In this study, glycation of human IgG was carried out with varying concentrations of glucose. Effect of incubation period on glycation of IgG has also been studied. Amadori adduct was detected by nitroblue tetrazolium (NBT) dye. The glucose mediated structural alterations in IgG were studied by UV, fluorescence, CD, FT-IR, DLS and DSC spectroscopy, and SDS-PAGE. Glycation-induced aggregation in AGE-IgG was reported in the form of binding of thioflavin T and congo red. Furthermore, AGE-modified IgG exhibited hyperchromicity, decrease of tryptophan fluorescence accompanied by increase in AGE specific fluorescence, loss of β-sheet, appearance of new peak in FT-IR, increase in hydrodynamic size and melting temperature. SDS-PAGE results showed decrease in the band intensity of glycosylated-IgG compared to native IgG. Glycation-induced modifications and aggregation of IgG might be important in the pathogenesis of RA.