Denaturation of proteins. IV. N.M.R. studies of ribonuclease-A

1972 ◽  
Vol 25 (1) ◽  
pp. 209 ◽  
Author(s):  
JH Bradbury ◽  
NLR King
Keyword(s):  
Formic Acid ◽  
Deuterium Oxide ◽  
Guanidine Hydrochloride ◽  
Difference Spectrum ◽  
Potassium Thiocyanate ◽  
Ribonuclease A ◽  
Relative Increase ◽  
Intermediate Form ◽  
State Process ◽  
Major Transition

The denaturation of ribonuclease-A by the addition of urea, guanidine hydrochloride, formic acid, and potassium thiocyanate to solutions in water or D2O at 33� has been followed by nuclear magnetic resonance (N.M.R.) spectroscopy. ��The complex N.M.R. spectra at low field can be simplified greatly by a difference spectrum obtained by subtracting the spectrum obtained in deuterium oxide from the corresponding spectrum in water, whence the resonances of protons attached to nitrogen are isolated. Binding of urea and guanidine hydrochloride at concentrations well below that needed for unfolding is shown by modification of the C2 histidine resonances due to the histidines located at positions 12 and 119. This confirms that these denaturants inactivate the enzyme by binding at its active site as proposed by Barnard.The unfolding of ribonuclease by urea and guanidine hydrochloride at acid pH is shown to be a two-state process in which the fraction of unfolded molecules (cross-linked random coils) is calculated directly from the relative increase in heights of the various n.m.r, resonances. The unfolding in [D2]formic acid is characterized by the first (major) transition with a midpoint at 8% [D2] formic acid (v/v) and a second (minor) transition centred at 58% [D2]formic acid. In pure formic acid there is evidence of aggregate formation. An intermediate form characterized by a double methionine SCH3 resonance occurs during the first transition. There are therefore a minimum of five different states present during this unfolding. The major unfolding process produced by potassium thiocyanate is followed by a refolding to a non-native ordered form. This unfolding process is incomplete and three different

Microscopic observation of the effect of some reagents on the physical state of casein

1960 ◽  
Vol 27 (3) ◽  
pp. 353-360 ◽  
Author(s):  
N. King
Keyword(s):  
Lactic Acid ◽  
Guanidine Hydrochloride ◽  
Sodium Dodecyl ◽  
Skim Milk ◽  
Potassium Thiocyanate ◽  
Bond Breaking ◽  
High Concentration ◽  
Casein Micelles ◽  
Visible Effect ◽  
Cheese Curd

SummaryObservations were made by anoptral phase-contrast microscopy of the effect of various reagents on the state of dispersion of casein micelles in skim-milk and in lactic acid and rennet coagula. Among the hydrogen-bond-breaking reagents, urea at higher (4M) concentration dispersed the casein micelles in skim-milk and in acid and rennet coagula. Sodium salicylate was effective at low (M) concentration. Guanidine hydrochloride at low concentration caused aggregation of the micelles in skim-milk but at high concentration caused dispersion, while at both concentrations it dispersed acid and rennet coagula. Lithium iodide and potassium thiocyanate flocculated the casein in skim-milk, but dispersed it in both types of coagula. Flocculation in skim-milk by these reagents was followed by slow coalescence of the casein.At substantially lower molar concentrations, sodium dodecyl sulphate, an anionic detergent, which attacks either the hydrophobic bonds or the salt linkages in proteins, dispersed micelles and both types of coagula. Sodium thioglycollate, a disulphide-bond-breaking reagent, had no visible effect on the casein.In rennet coagulum in which the calcium bridges have been destroyed by the sequestering action of EDTA or in lactic acid coagulum, urea at low concentrations caused a quick and almost complete coalescence of casein. Fibres were formed in the rennet coagulum when the coalesced material, which presumably consists of randomly coiled chains of unfolded casein macromolecules, was subjected to stretching or flowing. Similar chemical and physical conditions would appear to prevail when fibre formation takes place in cheddaring cheese curd.

Thermodynamic characterization of the partially denatured states of ribonuclease A in calcium chloride and lithium chloride

1985 ◽  
Vol 63 (10) ◽  
pp. 1058-1063 ◽  
Author(s):  
Faizan Ahmad
Keyword(s):  
Calcium Chloride ◽  
Lithium Chloride ◽  
Guanidine Hydrochloride ◽  
Thermodynamic Quantity ◽  
Ribonuclease A ◽  
Ultraviolet Region ◽  
Conformational Properties ◽  
Far Ultraviolet ◽  
Denatured States

The denaturations of ribonuclease A by calcium chloride and lithium chloride were studied by circular dichroism measurements in the far-ultraviolet region. The temperature dependence of the equilibrium constant for the unfolding of the protein by calcium chloride and lithium chloride gave values of 46 and 52 kcal mol−1 (1 cal = 4.1868 J) for the enthalpy of denaturation at 25 °C and pH 7.0, respectively. Thermodynamic parameters for the denaturation by calcium chloride and lithium chloride are compared with those for the heat and guanidine hydrochloride denaturation. It has been observed that the thermodynamic quantity, be it free energy, entropy, or enthalpy, cannot be related quantitatively to the extent of unfolding measured by various conformational properties of the protein.

scholarly journals Degradation of protein disulphide bonds in dilute alkali

1980 ◽  
Vol 189 (3) ◽  
pp. 507-520 ◽  
Author(s):  
T M Florence
Keyword(s):  
Equilibrium Constants ◽  
Guanidine Hydrochloride ◽  
Mercury Electrode ◽  
Stripping Voltammetry ◽  
Ribonuclease A ◽  
Initial Product ◽  
Cathodic Stripping Voltammetry ◽  
Disulphide Bonds ◽  
Protein Digests ◽  
Beta Elimination

The degradation of S–S bonds in 0.2 M-NaOH at 25 degrees C was studied for a series of proteins and simple aliphatic disulphide compounds, by using cathodic stripping voltammetry, ion-selective-electrode potentiometry, spectrophotometry and ultrafiltration. The disulphide bonds that dissociated in 0.2 M-NaOH were usually those that are solvent accessible and that can be reduced by mild chemical reductants. Some unexpected differences were found between similar proteins, both in the number of S–S bonds dissociated and in their rates of decomposition. Chymotrypsin has one S–S bond attacked, whereas chymotrypsinogen and trypsinogen have two. Ribonuclease A has two S–S bonds dissociated, but ribonuclease S and S-protein have three. Denaturation in 6 M-guanidine hydrochloride before alkaline digestion caused the loss of an additional S–S bond in ribonuclease A and insulin, and increased the rate of dissociation of the S–S bonds of some other proteins. The initial product of S–S bond dissociation in dilute alkali is believed to be a persulphide intermediate formed by a beta-elimination reaction. This intermediate is in mobile equilibrium with bisulphide ion, HS-, and decomposes at a mercury electrode or in acid solution to yield a stoichiometric amount of sulphide. Rate constants and equilibrium constants were measured for the equilibria between HS- and the intermediates involved in the alkaline dissociation of several proteins. Elemental sulphur was not detected in any of the protein digests. It is suggested that formation of HS- from a persulphide intermediate involves a hydrolysis reaction to yield a sulphenic acid derivative. The small polypeptides glutathione and oxytocin gave only a low yield of persulphide, and their alkaline decomposition must proceed by a mechanism different from that of the proteins.

Synthesis of Cyclic Nitramines From Products of the Cyclocondensation Reaction of Guanidine With 2,3,5,6-Tetrahydroxypiperazine-1,4-dicarbaldehyde

1994 ◽  
Vol 47 (11) ◽  
pp. 2033 ◽  
Author(s):  
IJ Dagley ◽  
JL Flippenanderson
Keyword(s):  
Nitric Acid ◽  
Hydrochloric Acid ◽  
Acetic Anhydride ◽  
Formic Acid ◽  
Guanidine Hydrochloride ◽  
Chloride Ion ◽  
Trifluoroacetic Anhydride ◽  
Cyclocondensation Reaction ◽  
Cyclic Nitramines ◽  
Cis Isomer

The reaction of 2,3,5,6-tetrahydroxypiperazine-1,4-dicarbaldehyde (1) with guanidine hydrochloride in hydrochloric acid can be controlled to give 2,6-diiminododecahydrodiimidazo[4,5-b:4′,5′-e] pyrazine (2a) or the cis isomer of 4,5-diamino-2-iminoimidazolidine (4). Compound (4) reacts with formaldehyde, or formic acid followed by reduction, to give 2-iminooctahydroimidazo[4,5-d] imidazole (7). Treatment of (2a) or (7) with nitric acid gives dinitro derivatives that were isolated as nitric acid salts of the cyclic guanidines. Reaction of the dinitro derivatives with nitric acid/acetic anhydride in the presence of chloride ion gives 4,8-dinitro-2,6-bis( nitroimino ) dodecahydrodiimidazo -[4,5-b:4′,5′-e] pyrazine (3a) and 1,3-dinitro-5-( nitroimino ) octahydroimidazo [4,5-d] imidazole (9). The reaction of (7) with nitric acid/ trifluoroacetic anhydride was controlled to give either the tetranitro or a dinitro bis ( trifluoroacetyl ) derivative of the corresponding bicyclic urea.

scholarly journals High-level expression and molecular characterization of a recombinant prolidase fromEscherichia coliNovaBlue

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5863 ◽  
Author(s):  
Tzu-Fan Wang ◽  
Meng-Chun Chi ◽  
Kuan-Ling Lai ◽  
Min-Guan Lin ◽  
Yi-Yu Chen ◽  
...  
Keyword(s):  
Guanidine Hydrochloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Negative Influence ◽  
Size Exclusion ◽  
State Process ◽  
Nickel Chelate ◽  
Exclusion Chromatography ◽  
Efficient Option ◽  
High Level

Long-term use of organophosphorus (OP) compounds has become an increasing global problem and a major threat to sustainability and human health. Prolidase is a proline-specific metallopeptidase that can offer an efficient option for the degradation of OP compounds. In this study, a full-length gene fromEscherichia coliNovaBlue encoding a prolidase (EcPepQ) was amplified and cloned into the commercially-available vector pQE-30 to yield pQE-EcPepQ. The overexpressed enzyme was purified from the cell-free extract of isopropyl thio-β-D-galactoside IPTG-inducedE. coliM15 (pQE-EcPepQ) cells by nickel-chelate chromatography. The molecular mass ofEcPepQ was determined to be about 57 kDa by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and the result of size-exclusion chromatography demonstrated that the enzyme was mainly present in 25 mM Tris–HCl buffer (pH 8.0) as a dimeric form. The optimal conditions forEcPepQ activity were 60 °C, pH 8.0, and 0.1 mM Mn2+ion. Kinetic analysis with Ala-Pro as the substrate showed that theKmandkcatvalues ofEcPepQ were 8.8 mM and 926.5 ± 2.0 s−1, respectively. The thermal unfolding ofEcPepQ followed a two-state process with one well-defined unfolding transition of 64.2 °C. Analysis of guanidine hydrochloride (GdnHCl)-induced denaturation by tryptophan emission fluorescence spectroscopy revealed that the enzyme had a [GdnHCl]0.5,N-Uvalue of 1.98 M. The purified enzyme also exhibited some degree of tolerance to various water/organic co-solvents. Isopropanol and tetrahydrofuran were very detrimental to the enzymatic activity ofEcPepQ; however, other more hydrophilic co-solvents, such as formamide, methanol, and ethylene glycol, were better tolerated. Eventually, the non-negative influence of some co-solvents on both catalytic activity and structural stability ofEcPepQ allows to adjust the reaction conditions more suitable forEcPepQ-catalyzed bioprocess.

pH dependence of the urea and guanidine hydrochloride denaturation of ribonuclease A and ribonuclease T1

Biochemistry ◽  
1990 ◽  
Vol 29 (10) ◽  
pp. 2564-2572 ◽  
Author(s):  
C. Nick Pace ◽  
Douglas V. Laurents ◽  
James A. Thomson
Keyword(s):  
Ph Dependence ◽  
Guanidine Hydrochloride ◽  
Ribonuclease A ◽  
Ribonuclease T1
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