DIFFERENTIAL STABILIZING EFFECTS OF BUFFERS ON STRUCTURAL STABILITY OF BOVINE SERUM ALBUMIN AGAINST UREA DENATURATION

The conformational stability of bovine serum albumin (BSA) against urea denaturation was investigated in aqueous solutions both in the absence and presence of buffers. Various buffers differing in polar and nonpolar characters such as sodium phosphate, Tris-HCl, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) HEPES and [3-(N-morpholino)propanesulfonic acid] MOPS buffers were used in this study. Urea-induced structural changes were analyzed using different probes, i.e., intrinsic fluorescence, ANS fluorescence and UV-difference spectral signal. Presence of different buffers in the incubation medium offered different degrees of resistance to the protein against urea-induced structural changes compared to those obtained in water (in the absence of buffers). A similar trend of buffer-induced structural resistance was noticed with three different probes. The stabilizing effect of these buffers followed the order: MOPS > HEPES > sodium phosphate > Tris-HCl > water. As found in MOPS and HEPES buffers, the highest stability of BSA can be attributed to the presence of morpholine and piperazine rings, respectively, in their structures. These groups might have produced a hydrophobic environment around the protein surface, thus stabilizing protein conformation against urea denaturation.

Free Radical Generation in Far-UV Synchrotron Radiation Circular Dichroism Assays—Protein and Buffer Composition Contribution

A useful tool to analyze the ligands and/or environmental contribution to protein stability is represented by the Synchrotron Radiation Circular Dichroism UV-denaturation assay that consists in the acquisition of several consecutive repeated far-UV SRCD spectra. Recently we demonstrated that the prevailing mechanism of this denaturation involves the generation of free radicals and reactive oxygen species (ROS). In this work, we analyzed the effect of buffering agents commonly used in spectroscopic measurements, including MOPS (3-(N-morpholino) propanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TRIS-HCl (tris-hydroxymethil aminomethane hydrochloride), and phosphate, on the efficiency of protein denaturation caused by exposure to UV radiation. Fluorescence experiments confirmed the presence of ROS and were used to determine the rate of ROS generation. Our results indicate that the efficiency of the denaturation process is strongly influenced by the buffer composition with MOPS and HEPES acting also as scavengers and that the presence of proteins itself influenced the ROS formation rate.

Determination of Solubility of 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic Acid and its Sodium Salt in Acetonitrile and Voltammetric Investigation of Sulphonamide Drugs in Different Solvents in Their Absence and Presence

Sulphonamide drugs (sulphamethazine, sulphamerazine, sulphadiazine, sulphathiazole) were studied in a wide potential window (between 2 and − 2 V) in acetonitrile, dimethyl sulphoxide and in 50–50 v/v% binary mixtures of acetonitrile and water. The voltammograms of the outlined compounds were very similar both in the anodic and cathodic part in each non-aqueous solvents except for sulphathiazole. These sulphonamide drugs were also investigated in presence of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and its sodium salt and the voltammograms changed due to an acid–base reaction. HEPES and its sodium salt could be investigated in acetonitrile only in their saturation concentration as they were slightly soluble in this solvent. In a separate experiment their solubilities were determined at 298 K in acetonitrile with the co-solvent calibration method using water as co-solvent. Complementary fluorescence studies in dimethyl sulphoxide did not show the presence of any interaction between sulphonamide drugs and HEPES as well as its sodium salt.

Polyamines Counteract Carbonate-Driven Proteasome Stalling in Alkaline Conditions

Cancer cells tend to increase intracellular pH and, at the same time, are known to intensively produce and uptake polyamines such as spermine. Here, we show that various amines, including biogenic polyamines, boost the activity of proteasomes in a dose-dependent manner. Proteasome activity in the classical amine-containing buffers, such as 2-(N-morpholino)ethanesulfonic acid (MES), Tris, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), glycylglycine, bis-Tris propane, and bicine, has a skewed distribution with a maximum at pH of 7.0–8.0. The activity of proteasomes in buffers containing imidazole and bis-Tris is maintained almost on the same level, in the pH range of 6.5–8.5. The third type of activation is observed in buffers based on the amino acids arginine and ornithine, as well as the natural polyamines spermine and spermidine. Proteasome activity in these buffers is dramatically increased at pH values greater than 7.5. Anionic buffers such as phosphate or carbonate, in contrast, inhibit proteasome activity during alkalization. Importantly, supplementation of a carbonate–phosphate buffer with spermine counteracts carbonate-driven proteasome stalling in alkaline conditions, predicting an additional physiological role of polyamines in maintaining the metabolism and survival of cancer cells.

Anion Effect on the Electropolymerization Reaction of Metanil Yellow in Aqueous Media and Characterization of Polymer Films

The electropolymerization of Metanil Yellow was investigated in aqueous solutions containing inorganic acids (sulphuric, hydrochloric, nitric, phosphoric and perchloric acid) as well as sulphonic acids (5-sulpho salicylic acid, sulphanilic acid, dodecylbenzene sulphonic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and lauryl sulphate in its acidic solution. By the inorganic ions the conductivity increased in the sulphate, chloride, nitrate, perchlorate serie. Of the organic sulphonic compounds 5-sulpho-salicylic acid was found to be a very efficient dopant in increasing polymer conductivity being obviously better than the other sulphonate anions. The formed conducting polymer was not suitable for detection of the corresponding anion. Nitrite ions completely diminished the electric properties of the polymer due to the reaction resulting nitrosamine.

Transient coating of γ-Fe2O3 nanoparticles with glutamate for its delivery to and removal from brain nerve terminals

Glutamate is the main excitatory neurotransmitter in the central nervous system and excessive extracellular glutamate concentration is a characteristic feature of stroke, brain trauma, and epilepsy. Also, glutamate is a potential tumor growth factor. Using radiolabeled ʟ-[14C]glutamate and magnetic fields, we developed an approach for monitoring the biomolecular coating (biocoating) with glutamate of the surface of maghemite (γ-Fe2O3) nanoparticles. The nanoparticles decreased the initial rate of ʟ-[14C]glutamate uptake, and increased the ambient level of ʟ-[14C]glutamate in isolated cortex nerve terminals (synaptosomes). The nanoparticles exhibit a high capability to adsorb glutamate/ʟ-[14C]glutamate in water. Some components of the incubation medium of nerve terminals, that is, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and NaH2PO4, decreased the ability of γ-Fe2O3 nanoparticles to form a glutamate biocoating by about 50% and 90%, respectively. Only 15% of the amount of glutamate biocoating obtained in water was obtained in blood plasma. Albumin did not prevent the formation of a glutamate biocoating. It was shown that the glutamate biocoating is a temporal dynamic structure at the surface of γ-Fe2O3 nanoparticles. Also, components of the nerve terminal incubation medium and physiological fluids responsible for the desorption of glutamate were identified. Glutamate-coated γ-Fe2O3 nanoparticles can be used for glutamate delivery to the nervous system or for glutamate adsorption (but with lower effectiveness) in stroke, brain trauma, epilepsy, and cancer treatment following by its subsequent removal using a magnetic field. γ-Fe2O3 nanoparticles with transient glutamate biocoating can be useful for multifunctional theranostics.

Modified HEPES one-pot synthetic strategy for gold nanostars

Gold nanostars are being used more regularly in the biosensing field. Despite their useful attributes, there is still a need to optimize aspects of the synthesis and stability. The seedless, synthetic method comprising 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) is a facile, rapid method; however, it produces heteromorphic nanostars. The modification of a HEPES method resulted in a silver-assisted, seedless gold nanostar synthesis method. The nanostars resulting from this method were monodispersed, multi-branched and approximately 37 ± 2 nm in diameter. It proved to be a repeatable method that produced homogeneous and robust nanostars. Once functionalized with polyvinylpyrrolidone 10 000, the new nanostars were observed to be stable in various environments such as salt, ionic strength and cell culture medium. In conclusion, the addition of the silver nitrate improved the morphology of the reported HEPES nanostars for the purpose of nanobiosensor development.

Retrospective analysis of efficacy of washed husband semen IUI for oligoasthenoteratospermia in an urban centre

Background: The true incidence of male subfertility is unknown due to great variability in the prevalence of subfertility. Artificial insemination with husband’s semen is the most widely used treatment for male infertility, usually presumed because of oligospermia, and for what is called ‘mucus hostility’ when there is failure of sperm penetration of cervical mucus despite normal seminal analysis.Methods: The study was conducted in 438 couples with male factor infertility at the ARTC (artificial reproductive technique centre) of Sri Ramakrishna Hospital, Coimbatore. Results of at least two seminograms (based on WHO norms) were used to primarily classify males into three categories-oligozoospermic, asthenozoo spermic and oligoasthenoteratospermic. The media used were the Earle’s Balanced Salt Solution (EBSS), Ham’s F10 and Medicult. EBSS and Ham’s F10 were obtained as “readymade” solutions from Sigma, USA. Medicult was imported from Denmark. EBSS and Ham’s F10 were supplemented with protein using FCS (Fetal cord Serum) or HEPES (4(2-hydroxyethyil)-1-piperazineethanesulfonic acid). Benzyl pencillin, 60mg per litre and Streptomycin, 50mg per litre were also to the media.Results: By the DMRT analysis of post wash count, the influence of the count below 5 million or above 20 million on the pregnancy rate was significant at all the levels of male factor.Conclusions: Considering the male factor, in cases of oligoasthenoteratospermia, IUI has a positive significant effect on the success rate of pregnancy at all three levels of the post wash sperm count.