muramidase and guanidine-thiocyanate

Protein aggregation and its subsequent deposition in different tissues culminate in a diverse range of diseases collectively known as amyloidoses. Aggregation of hen or human lysozyme depends on certain conditions, namely acidic pH or the presence of additives. In the present study, the effects on the aggregation of hen egg-white lysozyme via incubation in concentrated solutions of three different chaotropic agents namely guanidine thiocyanate, guanidine hydrochloride and urea were investigated. Here we used three different methods for the detection of the aggregates, thioflavin T fluorescence, circular dichroism spectroscopy and atomic force microscopy. Our results showed that upon incubation with different concentrations (0.5, 1.0, 2.0, 3.0, 4.0, 5.0M) of the chemical denaturants, lysozyme was aggregated at low concentrations of guanidine thiocyanate (1.0 and 2.0M) and at high concentrations of guanidine hydrochloride (4 and 5M), although no fibril formation was detected. In the case of urea, no aggregation was observed at any concentration.

Topics: Circular Dichroism; Guanidine; Guanidines; Microscopy, Atomic Force; Muramidase; Protein Binding; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Thiocyanates; Urea

The mechanism of denaturation and unfolding of lysozyme has been characterized here using the method of disulfide scrambling. Under denaturing conditions (urea, guanidinium hydrochloride (GdmCl), guanidinium thiocyanate (GdmSCN), or elevated temperature) and in the presence of thiol initiator, lysozyme denatures by shuffling its four native disulfide bonds and converts to a mixture of fully oxidized scrambled isomers. To denature 50% of the native lysozyme requires 1.1 M of GdmSCN, 2.8 M of GdmCl and 7.4 M of urea, respectively. High temperature (75 degrees C) denatures the native lysozyme quantitatively within 20 min. Analysis by reversed-phase high-performance liquid chromatography reveals that urea and GdmCl denatured lysozyme comprise a single predominant disulfide isomer, designated as X-lysozyme-a, regardless of the concentration of the denaturant. X-Lysozyme-a was shown to adopt the beads-form structure with its four disulfide bonds formed by four consecutive pairs of cysteines (Cys6-Cys30, Cys64-Cys76, Cys80-Cys94, Cys115-Cys127). The conspicuous absence of partially structured unfolding intermediates of lysozyme contrasts to that found in the case of alpha-lactalbumin and accounts for the widely observed two-stage mechanism of lysozyme unfolding.

Topics: Animals; Chickens; Chromatography, High Pressure Liquid; Disulfides; Enzyme Stability; Guanidine; Guanidines; Isomerism; Mercaptoethanol; Muramidase; Protein Denaturation; Protein Folding; Protein Renaturation; Protein Structure, Tertiary; Temperature; Thiocyanates; Urea

Topics: Bacterial Proteins; DNA, Bacterial; Electrophoresis, Agar Gel; Escherichia coli; Guanidines; Indicators and Reagents; Molecular Biology; Muramidase; Polyethylene Glycols; Ribonucleases; RNA, Bacterial; Sodium Dodecyl Sulfate; Thiocyanates

We have developed a novel plasmid isolation procedure and have adapted it for use on an automated nucleic acid extraction instrument. The protocol is based on the finding that phenol extraction of a 1 M guanidinium thiocyanate solution at pH 4.5 efficiently removes genomic DNA from the aqueous phase, while supercoiled plasmid DNA is retained in the aqueous phase. S1 nuclease digestion of the removed genomic DNA shows that it has been denatured, which presumably confers solubility in the organic phase. The complete automated protocol for plasmid isolation involves pretreatment of bacterial cells successively with lysozyme, RNase A, and proteinase K. Following these digestions, the solution is extracted twice with a phenol/chloroform/water mixture and once with chloroform. Purified plasmid is then collected by isopropanol precipitation. The purified plasmid is essentially free of genomic DNA, RNA, and protein and is a suitable substrate for DNA sequencing and other applications requiring highly pure supercoiled plasmid.

Topics: Automation; Detergents; DNA, Bacterial; Genetic Techniques; Guanidines; Hydrogen-Ion Concentration; Muramidase; Phenols; Plasmids; RNA, Bacterial; Sarcosine; Solutions; Thiocyanates