The modulation of structural stability of horseradish peroxidase as a consequence of macromolecular crowding.

Macromolecular crowding plays an inevitable role in all biological processes influencing association, conformation, and other characteristics of proteins. Present study is based on the effect of macromolecular crowding on structure of horseradish peroxidase (HRP) enzyme. Concentration-dependent conformational changes induced by crowding agents, dextran 70 and polyethylene glycol (PEG)-4000, were monitored employing a range of biophysical techniques. The intrinsic fluorescence spectra showed transition of protein from native to unfolded state. Marked increase in 8-Anilino-1-naphthalene-sulphonoic acid and Thioflavin T fluorescence indicated presence of non-native moieties with 80 mg/mL dextran. Enhanced absorbance in turbidity, Soret, and Congo red in corroboration with scattering intensity at 350nm results revealed incidence of HRP aggregates. A new peak around 218 nm in CD spectra pointed towards change in secondary structure towards ß-sheets. Significant loss of enzyme activity upon structural disruption was seen. Comet assay demonstrated DNA damage and genotoxic nature of HRP aggregates, supporting spectroscopic, and fluorescence results. The normalized results were obtained with 120 mg/mL PEG-4000 close to that of native HRP implying no disruptive effect on structure. It can be hypothesized that macromolecular crowding is a vital element, which can have diverse effects. In this study, dextran 70 was observed to have pro-aggregatory effect while enhanced stability of native enzyme was witnessed with PEG. Hence, it can be stated that PEG has potentially better crowder as it helps retain the native enzyme structure. Routine addition of crowding agents is recommended if biological molecules are to be studied under more physiologically appropriate environments.

Copper chloride inhibits brush border membrane enzymes, alters antioxidant and metabolic status and damages DNA in rat intestine: a dose-dependent study.

Copper (Cu) is an extensively used heavy metal and an indispensible micronutrient for living beings. However, Cu is also toxic and exerts multiple adverse health effects when humans are exposed to high levels of this metal. We have examined the effect of single acute oral dose of copper chloride (CuCl2) on parameters of oxidative stress, cellular metabolism, membrane and DNA damage in rat intestine. Adult male Wistar rats were divided into four groups and separately administered a single oral dose of 5, 15, 30 and 40 mg CuCl2/kg body weight. Rats not administered CuCl2 served as the control. Oral administration of CuCl2 led to significant alterations in the activities of metabolic and membrane-bound enzymes; brush border enzymes were inhibited by 45-75% relative to the control set. Inhibition of antioxidant enzymes diminished the metal-reducing and free radical quenching ability of the cells. Oxidative damage caused cellular oxidation of thiols, proteins and lipids. Diphenylamine and comet assays showed that CuCl2 treatment enhanced DNA damage while DNA-protein crosslinking was also increased in the intestinal cells. Examination of stained sections showed that CuCl2 treatment led to marked histological changes in the intestine. All the changes seen were in a CuCl2 dose-dependent manner with more prominent alterations at higher doses of CuCl2. These results clearly show that oral administration of CuCl2 results in oxidative damage to the intestine which can impair its digestive and absorptive functions.

Macromolecular crowding stabilises native structure of α-chymotrypsinogen-A against hexafluoropropanol-induced aggregates.

Cell interior is extremely congested with tightly packed biological macromolecules that exerts macromolecular crowding effect, influencing biophysical properties of proteins. To have a deeper insight into it we studied consequences of crowding on aggregation susceptibility and structural stability of α-chymotrypsinogen-A, pro-enzyme of serine protease family, upon addition of co-solvent reported to exert stress on polypeptides crafting favourable conditions for aggregation. Hexafluoropropan-2-ol (HFIP), a fluorinated alcohol caused structural disruption at 5% v/v unveiled by reduced intrinsic intensity and blue shifted ANS spectra. Significantly enhanced, red-shifted ThT and Congo red spectra sustained conformational changes concomitant with aggregation. FTIR and CD results confirmed transition of native structure to non-native extended, cross-linked beta-sheets. Transmission electron micrographs visibly exhibited incidence of amorphous aggregates. Macromolecular crowding, typically mimicked by concentrated solutions of dextran 70, was noticeably witnessed to defend conformational stability under denaturing condition. The native structure was retained maximally in presence of 100 mg/ml followed by 200 and 300 mg/ml dextran indicating concentration dependent deceleration of aggregate formation. It can be established that explicit consideration of crowding effects using relevant range of inert crowding agents must be a requisite for presumptions on intracellular conformational behaviour of proteins deduced from in vitro experiments.

Consequence of macromolecular crowding on aggregation propensity and structural stability of haemoglobin under glycating conditions.

Cell interiors are extremely congested with biological macromolecules exerting crowding effect, influencing various physiognomies of protein life. Present work deals with effect of crowding on folding behaviour of haemoglobin (Hb) under glycating conditions. Macromolecular crowding was mimicked by concentrated solutions of dextran 70. Hb with 0.2 M fructose and ribose was incubated separately for 96 h in dilute and crowded solution to analyse conformational changes. Reduced intrinsic and ANS fluorescence, decreased Soret absorbance, enhanced turbidity, browning of protein, red shift in ThT and Congo red spectra significantly unveiled protein aggregation. FTIR and CD results revealed transition from α-helix to ß-sheets confirming aggregation. Transmission electron microscopy exhibited incidence of aggregates. Macromolecular crowding was witnessed to defend conformational stability of native Hb under stress condition at 100 mg/ml dextran, noticeably indicating deceleration of aggregation. Stabilising effect of crowding was marginally better in fructosylated Hb than with ribose due to difference in their glycation potential. Contrarily, in over-crowded solution where dextran concentration was 500 mg/ml, heightened aggregation was perceived implying concentration dependant, dual nature of macromolecular crowding. The novelty of this study lies in idea of considering macromolecular crowding as a key player in regulation of protein stability which was safely ignored previously.

Carboxylic acids of different nature induces aggregation of hemoglobin.

Misfolded proteins that escape cellular quality control check lay the foundation for several progressively widespread neurodegenerative diseases, diabetes and others. Here, crotonic and citric acid are employed to study aggregation behaviour of hemoglobin (Hb). A systematic investigation on varying concentrations of acids from 0 to 60 mM on Hb gives an idea that transition is taking place in the vicinity of 10-30 mM. Hb showed increased intrinsic Trp fluorescence in the presence of both acids. A red shift of 10 nm in presence of citric acid contrary to a blue shift of 5 nm in presence of crotonic acid is observed. ANS and ThT fluorescence marked aggregation at 50 mM, supported by Congo red and Soret absorbance spectroscopy. CD, RLS and DLS studies also validate the findings. Molecular docking analysis exhibited the binding mode of Hb with acids. Aggregates were dense, beaded structure as visualised under TEM. Crotonic and citric acid at 20 and 30 mM, respectively, induced structural changes in Hb which transmutes to aggregate at higher concentration. These alterations remained almost constant and no significant changes were observed on increasing concentration further. Also, crotonic acid is more noxious, as it instigates conformational alterations at lower concentration than citric acid.