Activity and conformational changes of horseradish peroxidase in trifluoroethanol

2002 ◽  
Vol 80 (2) ◽  
pp. 205-213 ◽  
Author(s):  
Hong-Wei Zhou ◽  
Yan Xu ◽  
Hai-Meng Zhou
Keyword(s):  
Enzyme Activity ◽  
Horseradish Peroxidase ◽  
Conformational Changes ◽  
Tertiary Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Intrinsic Fluorescence ◽  
Two Phase ◽  
Main Effect ◽  
Α Helix ◽  
Kinetics Of

The effect of trifluoroethanol (TFE) on horseradish peroxidase (HRP) was determined using activity assay and spectral analysis including optical absorption, circular dichroism (CD), and intrinsic fluorescence. The enzyme activity increased nearly twofold after incubation with 5–25% (v/v) concentrations of TFE. At these TFE concentrations, the tertiary structure of the protein changed little, while small changes occurred at the active site. Further increases in the TFE concentration (25–40%) decreased the enzyme activity until at 40% TFE the enzyme was completely inactivated. The α-helix content of the protein increased at high TFE concentrations, while near-UV CD, Soret CD, and intrinsic fluorescence indicated that the tertiary structure was destroyed. Polyacrylamide gel electrophoresis results indicated that the surface charge of the enzyme was changed at TFE concentrations greater than 20%, and increasing concentrations of TFE reduced the enzyme molecular compactness. A scheme for the unfolding of HRP in TFE was suggested based on these results. The kinetics of absorption change at 403 nm in 40% TFE followed a two-phase course. Finally, HRP incubated with TFE was more sensitive to urea denaturation, which suggested that the main effect of TFE on HRP was the disruption of hydrophobic interactions.Key words: horseradish peroxidase, trifluoroethanol, unfolding, Soret.

Kinetic and structural analysis of the ultrasensitive behaviour of cyanobacterial ADP-glucose pyrophosphorylase

2000 ◽  
Vol 350 (1) ◽  
pp. 139-147 ◽  
Author(s):  
Diego F. GÓMEZ CASATI ◽  
Miguel A. AON ◽  
Alberto A. IGLESIAS
Keyword(s):  
Conformational Changes ◽  
Tertiary Structure ◽  
Fluorescence Emission ◽  
Maximal Activity ◽  
Size Exclusion ◽  
Exclusion Chromatography ◽  
Adp Glucose Pyrophosphorylase ◽  
Kinetics Of ◽  
Allosteric Regulators

The kinetic and (supra)molecular properties of the ultrasensitive behaviour of ADP-glucose pyrophosphorylase (AGPase) from Anabaena PCC 7120 (a cyanobacterium) were exhaustively studied. The response of the enzyme toward the allosteric activator 3-phosphoglycerate (3PGA) occurs with ultrasensitivity as a consequence of the cross-talk with the inhibitor Pi. Molecular ‘crowding’renders AGPase more sensitive to the interplay between the allosteric regulators and, consequently, enhances the ultrasensitive response. In crowded media, and when orthophosphate is present, the activation kinetics of the enzyme with 3PGA proceed with increased co-operativity and reduced affinity toward the activator. Under conditions of ultrasensitivity, the enzyme's maximal activation takes place in a narrow range of 3PGA concentrations. Moreover, saturation kinetics of the enzyme with respect to its substrates, glucose 1-phosphate and ATP, were different at low or high 3PGA levels in crowded media. Only under the latter conditions did AGPase exhibit discrimination between low or high levels of the activator, which increased the affinity toward the substrates and the maximal activity reached by the enzyme. Studies of fluorescence emission of tryptophan residues, fourth-derivative spectroscopy and size-exclusion chromatography indicated that the ultrasensitive behaviour is correlated with intramolecular conformational changes induced in the tertiary structure of the homotetrameric enzyme. The results suggest a physiological relevance of the ultrasensitive response of AGPase in vivo, since the enzyme could be subtly sensing changes in the levels of allosteric regulators and substrates, and thus determining the flux of metabolites toward synthesis of storage polysaccharides.

scholarly journals Superactivity and conformational changes on alpha-chymotrypsin upon interfacial binding to cationic micelles

2004 ◽  
Vol 378 (3) ◽  
pp. 1059-1066 ◽  
Author(s):  
M. Soledad CELEJ ◽  
Mariana G. D'ANDREA ◽  
Patricia T. CAMPANA ◽  
Gerardo D. FIDELIO ◽  
M. Lucia BIANCONI
Keyword(s):  
Conformational Changes ◽  
Tertiary Structure ◽  
Enzyme Stability ◽  
Catalytic Efficiency ◽  
Tryptophan Fluorescence ◽  
Enzyme Activation ◽  
Hexadecyltrimethylammonium Bromide ◽  
Negative Band ◽  
Cationic Micelles ◽  
Α Helix

The catalytic behaviour of α-CT (α-chymotrypsin) is affected by cationic micelles of CTABr (hexadecyltrimethylammonium bromide). The enzyme–micelle interaction leads to an increase in both the Vmax and the affinity for the substrate p-nitrophenyl acetate, indicating higher catalytic efficiency for bound α-CT. The bell-shaped profile of α-CT activity with increasing CTABr concentrations suggests that the micelle-bound enzyme reacts with the free substrate. Although more active with CTABr micelles, the enzyme stability is essentially the same as observed in buffer only. Enzyme activation is accompanied by changes in α-CT conformation. Changes in tertiary structure were observed by the increase in intensity and the red shift in the α-CT tryptophan fluorescence spectrum, suggesting the annulment of internal quenching and a more polar location of tryptophan residues. Near-UV CD also indicated the transfer of aromatic residues to a more flexible environment. CTABr micelles also induces an increase in α-helix, as seen by far-UV CD and FTIR (Fourier-transform infrared) spectroscopies. The far-UV CD spectrum of α-CT shows an increase in the intensity of the positive band at 198 nm and in the negative band at 222 nm, indicating an increased α-helical content. This is in agreement with FTIR studies, where an increase in the band at 1655 cm−1, corresponding to the α-helix, was shown by fitting analysis and difference spectroscopy. Spectral deconvolution indicated a reduction in the β-sheet content in micelle-bound α-CT. Our data suggest that the higher catalytic efficiency of micelle-bound α-CT results from significant conformational changes.

Inactivation and conformational changes of creatine kinase at low concentrations of hexafluoroisopropanol solutions

2003 ◽  
Vol 81 (5) ◽  
pp. 327-333 ◽  
Author(s):  
Xiao-Yun Wang ◽  
Fan-Guo Meng ◽  
Hai-Meng Zhou
Keyword(s):  
Creatine Kinase ◽  
Enzyme Activity ◽  
Conformational Changes ◽  
Fluorescence Spectra ◽  
Cd Spectra ◽  
Irreversible Inhibition ◽  
Low Concentrations ◽  
Flexible Region ◽  
Far Ultraviolet ◽  
Kinetics Of

Using the methods of far-ultraviolet circular dichroism (CD) spectra, fluorescence spectra, and enzyme activity assays, the inactivation and conformational changes of creatine kinase (CK) induced by 1,1,1,3,3,3-hexafluoro-2-propanol (hexafluoroisopropanol (HFIP)) of different concentrations were investigated. To avoid the aggregation of CK that occurs with high HFIP, concentrations of 0%–5% HFIP were used in this study. The CD spectra showed that HFIP concentrations above 2.5% strongly induced the formation of secondary structures of CK. No marked conformational changes were observed at low concentrations of HFIP (0%–2.5%). After incubation with 0.2% HFIP for 10 min, CK lost most of its activity. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity described previously by Tsou was applied to study the kinetics of CK inactivation during denaturation by HFIP. The inactivation rate constants for the free enzyme and the substrate–enzyme complex were determined by Tsou's method. The results suggested that low concentrations of HFIP had a high potential to induce helices of protein and that the active site of the enzyme was situated in a limited and flexible region of the enzyme molecule that was more susceptible to the denaturant than was the protein as a whole.Key words: creatine kinase, inactivation, conformation, kinetics, hexafluoroisopropanol.

scholarly journals Inactivation kinetics of dihydrofolate reductase from Chinese hamster during urea denaturation

1997 ◽  
Vol 324 (2) ◽  
pp. 395-401 ◽  
Author(s):  
Jia-Wei WU ◽  
Zhi-Xin WANG ◽  
Jun-Mei ZHOU
Keyword(s):  
Enzyme Activity ◽  
Dihydrofolate Reductase ◽  
Single Phase ◽  
Tertiary Structure ◽  
Chinese Hamster ◽  
Free Enzyme ◽  
Inactivation Kinetics ◽  
Binary And Ternary Complexes ◽  
Enzyme Substrate ◽  
Kinetics Of

The kinetic theory of substrate reaction during modification of enzyme activity has been applied to the study of inactivation kinetics of Chinese hamster dihydrofolate reductase by urea [Tsou (1988) Adv. Enzymol. Relat. Areas Mol. Biol. 61, 381–436]. On the basis of the kinetic equation of substrate reaction in the presence of urea, all microscopic kinetic constants for the free enzyme and enzyme–substrate binary and ternary complexes have been determined. The results of the present study indicate that the denaturation of dihydrofolate reductase by urea follows single-phase kinetics, and changes in enzyme activity and tertiary structure proceed simultaneously in the unfolding process. Both substrates, NADPH and 7,8-dihydrofolate, protect dihydrofolate reductase against inactivation, and enzyme–substrate complexes lose their activity less rapidly than the free enzyme.

scholarly journals The Conformational Structural Change of Soy Glycinin via Lactic Acid Bacteria Fermentation Reduced Immunoglobulin E Reactivity

Foods ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 2969
Author(s):  
Zhen Liu ◽  
Yaqiong Wang ◽  
Yifei Liu ◽  
Qiuqin Zhang ◽  
Wei Li ◽  
...  
Keyword(s):  
Particle Size ◽  
Conformational Changes ◽  
Fluorescence Intensity ◽  
Immunoglobulin E ◽  
Principal Component ◽  
Surface Hydrophobicity ◽  
Intrinsic Fluorescence ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ige Reactivity ◽  
Α Helix

This study investigated the fermentation of isolated soy glycinin by using the Lactiplantibacillus plantarum B1-6 strain, its reduction effect on immunoglobulin E (IgE) reactivity, the relationship with protein aggregation/gelation state and conformational changes. Fermentation was performed under different glycinin concentrations (0.1%, 0.5%, 1% and 2%, w/v) and varied fermentation terminal pH levels (FT-pH) (pH 6.0, 4.5, 4.0 and 3.5). L. plantarum B1-6 showed potency in reducing immunoreactivity to 0.10–69.85%, as determined by a sandwich enzyme-linked immunosorbent assay. At a FT-pH of 6.0 and 4.5, extremely low IgE reactivity (0.1–22.32%) was observed. Fermentation resulted in a great increase (2.31–6.8-fold) in particle size and a loss of intensity in A3 and basic subunits. The conformation of glycinin was altered, as demonstrated by improved surface hydrophobicity (1.33–7.39-fold), decreased intrinsic fluorescence intensity and the α-helix structure. Among the four selected concentrations, glycinin at 1% (w/v, G-1) evolved the greatest particles during fermentation and demonstrated the lowest immunoreactivity. Principal component analysis confirmed that particle size, intrinsic fluorescence intensity, α-helix and ionic bond were closely related to immunoreactivity reduction.

scholarly journals A Spectroscopic Study on PtCl4(2−) Binding to Rabbit Skeletal Muscle G-Actin

1995 ◽  
Vol 2 (3) ◽  
pp. 127-136 ◽  
Author(s):  
Juan Zou ◽  
Hong-Ye Sun ◽  
Kui Wang
Keyword(s):  
Conformational Changes ◽  
Binding Sites ◽  
Binding Constant ◽  
Intrinsic Fluorescence ◽  
Molar Ratio ◽  
Second Step ◽  
High Affinity ◽  
Α Helix ◽  
Discontinuous Mode ◽  
Sulfur Containing

It was found that the binding of PtCl42− to G-actin and the consequent conformational changes are different with those for hard acids. It is a two-step process depending on molar ratio PtCl42−/actin (R). In the first step, R less than 25, the PtCl42− ions are bound to sulfur-containing groups preferentially. These high-affinity sites determined by Scatchard approach are characterized by n1 = 30 with average binding constant K1=1.0×107M-1. The conformational changes are significant as characterized by N-(1-pyrenyl) maleimide(NPM) labeled fluorescence, intrinsic fluorescence and CD spectra. EPR spectroscopy of maleimide spin labeled(MSL) actin demonstrated that even PtCl42−binding is limited to a very small fraction of high-affinity sites(R<1), it can bring about a pronounced change of conformation. In the range of R=25-40, high-affinity sites accessible are saturated. In the second step(R>40) , deep-buried binding sites turn out to be accessible as a result of the accumulated conformational changes. These new binding sites are estimated to be n2=26 with average binding constant K2=2.1×106M-1. Although in this step the quenching of intrinsic fluorescence goes on and the NPM-labled thiols moves to more hydrophilic environment, no change in α-helix content was found. These results suggested that with increasing in PtCl42− binding, the G-actin turns to an open and loose structure in a discontinuous mode.

Platelet Microtubule Subunit Proteins

1979 ◽  
Vol 42 (05) ◽  
pp. 1630-1633 ◽  
Author(s):  
A G Castle ◽  
N Crawford
Keyword(s):  
Epithelial Cells ◽  
Gel Electrophoresis ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Lens Epithelial Cells ◽  
Molecular Weights ◽  
Kinetics Of ◽  
Microtubular Structures

SummaryBlood platelets contain microtubule proteins (tubulin and HMWs) which can be polymerised “in vitro” to form structures which resemble the microtubules seen in the intact platelet. Platelet tubulin is composed of two non-identical subunits a and p tubulin which have molecular weights around 55,000 but can be resolved in alkaline SDS-polyacrylamide gel electrophoresis. These subunits associate as dimers with sedimentation coefficients of about 5.7 S although it is not known whether the dimer protein is a homo- or hetero-dimer. The dimer tubulin binds the anti-mitotic drug colchicine and the kinetics of this binding are similar to those reported for neurotubulins. Platelet microtubules also contain two HMW proteins which appear to be essential and integral components of the fully assembled microtubule. These proteins have molecular weights greater than 200,000 daltons. Fluorescent labelled antibodies to platelet and brain tubulins stain long filamentous microtubular structures in bovine lens epithelial cells and this pattern of staining is prevented by exposing the cells to conditions known to cause depolymerisation of cell microtubules.

Interphase distribution of 2-furylethylenes

1985 ◽  
Vol 50 (8) ◽  
pp. 1642-1647 ◽  
Author(s):  
Štefan Baláž ◽  
Anton Kuchár ◽  
Ernest Šturdík ◽  
Michal Rosenberg ◽  
Ladislav Štibrányi ◽  
...  
Keyword(s):  
Partition Coefficient ◽  
Partition Coefficients ◽  
Transport Rate ◽  
Phase System ◽  
Two Phase ◽  
Interphase Distribution ◽  
Distribution Kinetics ◽  
System 1 ◽  
Kinetics Of

The distribution kinetics of 35 2-furylethylene derivatives in two-phase system 1-octanol-water was investigated. The transport rate parameters in direction water-1-octanol (l1) and backwards (l2) are partition coefficient P = l1/l2 dependent according to equations l1 = logP - log(βP + 1) + const., l2 = -log(βP + 1) + const., const. = -5.600, β = 0.261. Importance of this finding for assesment of distribution of compounds under investigation in biosystems and also the suitability of the presented method for determination of partition coefficients are discussed.

scholarly journals Soluble Expression and Efficient Purification of Recombinant Class I Hydrophobin DewA

2021 ◽  
Vol 22 (15) ◽  
pp. 7843
Author(s):  
Sang-Oh Ahn ◽  
Ho-Dong Lim ◽  
Sung-Hwan You ◽  
Dae-Eun Cheong ◽  
Geun-Joong Kim
Keyword(s):  
Tertiary Structure ◽  
Signal Sequence ◽  
Soluble Expression ◽  
Class I ◽  
Soluble Form ◽  
Two Phase ◽  
E Coli ◽  
Small Proteins ◽  
Industrial Use ◽  
Shed Light

Hydrophobins are small proteins (<20 kDa) with an amphipathic tertiary structure that are secreted by various filamentous fungi. Their amphipathic properties provide surfactant-like activity, leading to the formation of robust amphipathic layers at hydrophilic–hydrophobic interfaces, which make them useful for a wide variety of industrial fields spanning protein immobilization to surface functionalization. However, the industrial use of recombinant hydrophobins has been hampered due to low yield from inclusion bodies owing to the complicated process, including an auxiliary refolding step. Herein, we report the soluble expression of a recombinant class I hydrophobin DewA originating from Aspergillus nidulans, and its efficient purification from recombinant Escherichia coli. Soluble expression of the recombinant hydrophobin DewA was achieved by a tagging strategy using a systematically designed expression tag (ramp tag) that was fused to the N-terminus of DewA lacking the innate signal sequence. Highly expressed recombinant hydrophobin DewA in a soluble form was efficiently purified by a modified aqueous two-phase separation technique using isopropyl alcohol. Our approach for expression and purification of the recombinant hydrophobin DewA in E. coli shed light on the industrial production of hydrophobins from prokaryotic hosts.

Separation and purification of horseradish peroxidase from horseradish roots using a novel integrated method

2021 ◽  
Author(s):  
Wei Ji ◽  
Wenmei Ao ◽  
Mengqiu Sun ◽  
Chunlai Feng ◽  
Yun Wang
Keyword(s):  
Horseradish Peroxidase ◽  
Phase Extraction ◽  
Separation And Purification ◽  
Two Phase ◽  
Affinity Precipitation ◽  
Integrated Method ◽  
Novel Method ◽  
Temperature Controlled ◽  
Aqueous Two Phase Extraction

The aim of the present work was to develop a novel method integrating two-step aqueous two-phase extraction and temperature-controlled affinity precipitation for the separation and purification horseradish peroxidase (HRP) from...

Sign in / Sign up

Export Citation Format

Share Document