scholarly journals Denaturation of proteins and nucleic acids by thermal-gradient electrophoresis

1981 ◽  
Vol 197 (1) ◽  
pp. 105-109 ◽  
Author(s):  
D R Thatcher ◽  
B Hodson
Keyword(s):  
Conformational Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Complex Mixture ◽  
Effect Of Temperature ◽  
Electrophoretic Method ◽  
Denatured State ◽  
Double Stranded Dna ◽  
Electrophoresis Method ◽  
Dna Restriction ◽  
Irreversible Unfolding

A polyacrylamide-gel-electrophoresis method has been developed that permits the analysis of conformational changes that occur during the thermal denaturation of macromolecules. A stable transverse temperature gradient was produced in an aluminium heating jacket clamped around a vertical polyacrylamide slab gel. After temperature equilibration, gels were loaded with either a layer of protein solution (20-200 micrograms/gel) or a solution of double-stranded DNA (20 micrograms/gel) and electrophoresis begun. At the end of the run the gels were stained and the effect of temperature on mobility observed. The technique proved informative both for the irreversible unfolding of proteins (Drosophila alcohol dehydrogenase and lactic acid dehydrogenase) and for a protein that was reversibly denatured by heat (beta-lactamase). In the latter case a clear transition between the native enzyme and a slower-migrating denatured state was observed. The patterns obtained were analogous to the type produced by the transverse-urea-gradient-electrophoretic method of Creighton [(1979) J. Mol. Biol. 129, 253-264]. The method also resolved a complex mixture of double-stranded-DNA restriction-digest fragments.

scholarly journals The Native Monomer of Bacillus Pumilus Ribonuclease Does Not Exist Extracellularly

2018 ◽  
Vol 2018 ◽  
pp. 1-7 ◽  
Author(s):  
Olga Ilinskaya ◽  
Vera Ulyanova ◽  
Irina Lisevich ◽  
Elena Dudkina ◽  
Nataliya Zakharchenko ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Protein Concentration ◽  
Bacillus Pumilus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Culture Fluid ◽  
Size Exclusion ◽  
High Protein Concentration ◽  
Exclusion Chromatography ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Hypochromic Effect

Supported by crystallography studies, secreted ribonuclease of Bacillus pumilus (binase) has long been considered to be monomeric in form. Recent evidence obtained using native polyacrylamide gel electrophoresis and size-exclusion chromatography suggests that binase is in fact dimeric. To eliminate ambiguity and contradictions in the data we have measured conformational changes, hypochromic effect, and hydrodynamic radius of binase. The immutability of binase secondary structure upon transition from low to high protein concentration was registered, suggesting the binase dimerization immediately after translocation through the cell membrane and leading to detection of binase dimers only in the culture fluid regardless of ribonuclease concentration. Our results made it necessary to take a fresh look at the binase stability and cytotoxicity towards virus-infected or tumor cells.

Study on Degradation of Radix Isatidis Polypeptide in Artificial Gastrointestinal Environment

2014 ◽  
Vol 989-994 ◽  
pp. 1020-1024
Author(s):  
Nan Nan ◽  
Xi Jing Liu
Keyword(s):  
Gel Electrophoresis ◽  
Free Amino ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Radix Isatidis ◽  
Sds Page ◽  
Electrophoresis Method ◽  
Amino Acid Levels ◽  
Different Molecular Weight

Radix Isatidis is a traditional Chinese medicine for treatment of influenza and inflammation in China. In this paper, in order to study the degradation situation of Radix Isatidis polypeptide in artificial gastrointestinal environment, the SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) method was used to detect the degradation of Radix Isatidis polypeptide in artificial intestinal juice and gastric juice, and it showed that Radix Isatidis peptides could be degradated to different degrees. HPLC (High Performance Liquid Chromatography) was used to determine the change of peptides degradation, and it indicated that free amino acid levels did not change significantly. The result after degradation was also detected by BCA method, and it showed that there were still a large number of polypeptides in the liquid. From this experiment we can come to this conclusion that Radix Isatidis polypeptides in artificial gastrointestinal juice mostly degraded into a series of different molecular weight peptides.

Polyacrylamide gel electrophoresis of exoenzymes produced by Pseudomonas fluorescens strain W

1971 ◽  
Vol 17 (2) ◽  
pp. 241-248 ◽  
Author(s):  
Harvey Winters ◽  
W. A. Corpe
Keyword(s):  
Pseudomonas Fluorescens ◽  
Bacterial Cell ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Complex Mixture ◽  
Gram Negative ◽  
Coomassie Blue ◽  
The Individual ◽  
Cell Envelopes

Culture filtrates from strain W of Pseudomonas fluorescens that cause lysis of gram-negative bacterial cell envelopes were examined for specific hydrolases. The enzymes were concentrated by ammonium sulfate fractionation and by column fractionation on Sephadex G-100. Attempts to separate the individual hydrolases quantitatively by elution from DEAE-cellulose failed because of the formation of aggregates. Resolution of the individual hydrolases was accomplished by disc gel electrophoresis using polyacrylamide (7.5%) gels buffered at pH 8.9 with Tris-glycine. The enzyme mixture was separated into 13 distinct protein bands which were stained with Coomassie blue. The individual hydrolases were detected either directly on the gels or by assay after elution from gel segments and included four proteinases, three phosphatases, two β-glucosidases, one ribonuclease, one lipase, one esterase, and one catalase. These methods provide a rapid, sensitive technique for the detection of many individual hydrolases in a complex mixture.

scholarly journals Structure and genome release of Twort-like Myoviridae phage with a double-layered baseplate

2016 ◽  
Vol 113 (33) ◽  
pp. 9351-9356 ◽  
Author(s):  
Jiří Nováček ◽  
Marta Šiborová ◽  
Martin Benešík ◽  
Roman Pantůček ◽  
Jiří Doškař ◽  
...  
Keyword(s):  
Cell Wall ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Phage Genome ◽  
Protein Complexes ◽  
Protein Subunit ◽  
Double Stranded Dna ◽  
Cryo Electron Microscopy ◽  
Tail Sheath ◽  
The Family

Bacteriophages from the family Myoviridae use double-layered contractile tails to infect bacteria. Contraction of the tail sheath enables the tail tube to penetrate through the bacterial cell wall and serve as a channel for the transport of the phage genome into the cytoplasm. However, the mechanisms controlling the tail contraction and genome release of phages with “double-layered” baseplates were unknown. We used cryo-electron microscopy to show that the binding of the Twort-like phage phi812 to the Staphylococcus aureus cell wall requires a 210° rotation of the heterohexameric receptor-binding and tripod protein complexes within its baseplate about an axis perpendicular to the sixfold axis of the tail. This rotation reorients the receptor-binding proteins to point away from the phage head, and also results in disruption of the interaction of the tripod proteins with the tail sheath, hence triggering its contraction. However, the tail sheath contraction of Myoviridae phages is not sufficient to induce genome ejection. We show that the end of the phi812 double-stranded DNA genome is bound to one protein subunit from a connector complex that also forms an interface between the phage head and tail. The tail sheath contraction induces conformational changes of the neck and connector that result in disruption of the DNA binding. The genome penetrates into the neck, but is stopped at a bottleneck before the tail tube. A subsequent structural change of the tail tube induced by its interaction with the S. aureus cell is required for the genome’s release.

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.

scholarly journals Microbial Conversion of Shrimp Heads to Proteases and Chitin as an Effective Dye Adsorbent

Polymers ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 2228 ◽  
Author(s):  
Chien Thang Doan ◽  
Thi Ngoc Tran ◽  
Chuan-Lu Wang ◽  
San-Lang Wang
Keyword(s):  
Culture Media ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protease Production ◽  
Adsorptive Capacity ◽  
Microbial Conversion ◽  
N Nutrition ◽  
Chitin Extraction ◽  
Electrophoresis Method ◽  
Shrimp Head ◽  
Shrimp Heads

As a green and effective technique in the production of a large number of valuable products, the microbial conversion of chitinous fishery wastes is receiving much attention. In this study, protease production using the Paenibacillus mucilaginosus TKU032 strain was conducted on culture media containing several common types of chitinous fishery by-products serving as the carbon and nitrogen (C/N) nutrition source. Among the chitinous wastes, 1.5% (w/v) shrimp head powder (SHP) was found to be the most appropriate nutritional source for protease production when a maximal enzyme activity of 3.14 ± 0.1 U/mL was observed on the 3rd day of the culture period. The molecular mass of P. mucilaginosus TKU032 protease was estimated to be nearly 32 kDa by the polyacrylamide gel electrophoresis method. The residual SHP obtained from the culture medium was also considered to be utilized for chitin extraction. The deproteinization rate of the fermentation was estimated to be 45%, and the chitin obtained from fermented SHP (fSHP) displayed a similar characteristic Fourier-transform infrared spectroscopy (FTIR) profile as that from SHP. In addition, SHP, fSHP, and chitins obtained from SHP and fSHP were investigated for their adsorptive capacity of nine types of dyes, and chitin obtained from fSHP displayed a good adsorption rate on Congo Red and Red No. 7, at 99% and 97%, respectively. In short, the results provide potential support for the utilization of SHP in the production of P. mucilaginosus TKU032 protease via the fermentation as well as the preparation of chitin from fSHP as an effective dye adsorbent.

Comparison of the effect of poly(N-vinyl caprolactam) and poly(N-isopropyl acrylamide) trimers on the stability of hydrated Na-montmorillonite: A molecular dynamics study

2020 ◽  
pp. 096739112093524
Author(s):  
Jiafang Xu ◽  
Moussa Camara ◽  
Hualin Liao ◽  
Hong Guo ◽  
Kouassi Louis Kra ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Conformational Changes ◽  
Stable State ◽  
Dynamics Simulation ◽  
Effect Of Temperature ◽  
Radius Of Gyration ◽  
Molecular Arrangement ◽  
Isopropyl Acrylamide ◽  
The Stability ◽  
The Impact

In the present study, we performed a molecular dynamics simulation of the intercalation of poly( N-isopropyl acrylamide) (NIPAM)3 and poly( N-vinyl caprolactam) (NVCL)3 trimers into Na-montmorillonite (Na-Mt) to evaluate their effects on the interlayer structure and the stability of hydrated Na-Mt. The impact of both trimers on the interlayer species and their dynamics properties at different temperatures in a canonical ensemble (NVT) were investigated. The results showed that the electrostatic forces exerted by Na cations on H2O molecules and the interlayer H2O molecular arrangement are not affected by the rise in temperature after adding both trimers. Trimer addition reinforced the structure of interlayer H2O molecules so that the effect of temperature increase on them became negligible. The structural dynamics evolution of the radius of gyration of both trimers showed the existence of conformation changes when temperature increased. These conformational changes are more complex in the case of (NVCL)3 than (NIPAM)3 due to its large monomers. Both trimers reduced the mobility of interlayer particles with a better inhibition effect obtained for (NVCL)3 compared to (NIPAM)3. The concentration profile of interlayers’ species showed the affinity of Na cations for clay mineral surfaces while H2O molecules moved away. Compared these two trimers, the most stable state of Na-Mt is achieved with (NVCL)3. These results could help highlight the inhibition properties of (NIPAM)3 and (NVCL)3 on hydrated Na-Mt and to predict its stability against changes in environmental conditions.

scholarly journals Purification and properties of β-mannanases I and II from the germinated seeds of Trifolium repens. Mode of galactomannan degradation in vitro

1978 ◽  
Vol 175 (3) ◽  
pp. 1079-1087 ◽  
Author(s):  
H Villarroya ◽  
J Williams ◽  
P Dey ◽  
S Villarroya ◽  
F Petek
Keyword(s):  
Trifolium Repens ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Disc Electrophoresis ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Electrophoretic Method ◽  
Germinated Seeds

Two beta-mannanases (beta-mannosidases, EC 3.2.1.25) purified from the germinated seeds of Trifolium repens by a procedure that included chromatography on hydroxyapatite, gel filtration on acrylamide/agarose (Ultragel 5/4) and preparative polyacrylamide-gel-electrophoresis. The final purification step completely resolved two beta-mannanases with distinct specificities, which were termed beta-mannanase I and beta-mannanase II. beta-Mannanase I was purified 1400-fold and beta-mannanase II 1000-fold. The purified enzymes showed a single protein band when examined by polyacrylamide-gel disc electrophoresis. beta-Mannanase I, apparent mol.wt. 43 000, accounted for 49% of the total activity recovered from the final step of purification. beta-Mannanase II, apparent mol.wt. 38 000, accounted for the remaining 51% of activity. Molecular-weight determinations were by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by the electrophoretic method of Hendrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164]. The substrate specificities of both enzymes were examined with the galactomannans of T. repens and of Medicago sativa, as well as with manno-oligosaccharides. The pH optimum was between pH 5.1 and 5.6 for both enzymes.

Identification of Apo(a) Phenotypes in a Korean Population Using a Standardized Nomenclature System Based on the Number of Kringle IV Repeats

1997 ◽  
Vol 34 (6) ◽  
pp. 681-687
Author(s):  
Won-Ki Min ◽  
Jae Ok Lee ◽  
Chun Hee Kim ◽  
Junghan Song ◽  
Jin Q Kim
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Serum Lipoprotein ◽  
Allele Size ◽  
Nomenclature System ◽  
Molecular Weights ◽  
Apolipoprotein A ◽  
Electrophoresis Method

We determined serum lipoprotein(a) [Lp(a)] concentrations and apolipoprotein(a) [apo(a)] phenotypes in 193 healthy Koreans. We analysed the apo(a) phenotypes by a simplified sodium dodecyl sulphate-polyacrylamide gel electrophoresis method and classified apo(a) isoforms objectively using an apo(a) phenotype standard with a known number of kringle IV repeats. The frequency distribution of Lp(a) levels showed marked positive skew with a mean of 0·143 g/L and a median of 0·052 g/L. Of the 193 subjects tested, no bands were detected in three, and single- and double-band phenotypes were observed in 103 and 87, respectively. Among the Koreans, the most frequent phenotype was S5(39·4%), followed by S4S5(17·1%), S5S5(14·0%), S4(11·4%), S3S5(5·2%), and the remaining phenotypes (13·0%). The calculated apo(a) allele frequencies were LpF, 0·003; LpS1, 0·013; LpS2, 0·010; LpS3 0·048; LpS4 0·198; LpS5, 0·563 and Lp0, 0·165. We found that the serum Lp(a) concentration in Koreans was similar to that of Caucasians, but the apo(a) allele size distribution was shifted toward higher molecular weights.

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