scholarly journals A fluorimetric study of the role of calcium ions in the stability of thermolysin

1977 ◽  
Vol 165 (3) ◽  
pp. 539-545 ◽  
Author(s):  
Angelo Fontana ◽  
Claudio Vita ◽  
Enrico Boccu' ◽  
Francesco M. Veronese
Keyword(s):  
Calcium Ions ◽  
Fluorescence Emission ◽  
Protein Molecule ◽  
Fluorescence Emission Spectrum ◽  
Guanidinium Chloride ◽  
Emission Maximum ◽  
Heat Stable ◽  
Tryptophan Residues ◽  
The Stability

1. Fluorimetric techniques were used to characterize the environment of tryptophan residues in thermolysin and apo-thermolysin. The apo-thermolysin was obtained by dissolving the enzyme in the presence of 10mm-EDTA, which removed the functional Zn2+ ion and the four Ca2+ ions/molecule from the enzyme. 2. At 25°C in aqueous solution the fluorescence-emission spectrum of the native holoenzyme, on excitation at 290nm, was essentially characteristic of tryptophan, with an emission maximum at 333nm. The emission maximum of the apoenzyme is red-shifted to 338nm and the relative intensity of fluorescence is decreased by 10%, both effects indicating some unfolding of the protein molecule, with the indole groups being transferred to a more hydrophilic environment. 3. Fluorescence quenching studies using KI, N′-methylnicotinamide hydrochloride and acrylamide indicated a more open structure in the apoenzyme, with the tryptophan residues located in a negatively charged environment. 4. The thermal properties of the apoenzyme, as monitored by fluorescence-emission measurements, are dramatically changed with respect to the native holoenzyme. In fact, whereas the native enzyme is heat-stable up to about 80°C, for the apoenzyme a thermal transition is observed near 48°C. The apoenzyme is also unstable to the action of unfolding agents such as urea and guanidinium chloride, much as for other globular proteins from mesophilic organisms. 5. The functional Zn2+ ion does not contribute noticeably to the stability of thermolysin. 6. It is concluded that a major role in the structural stability of thermolysin is played by the Ca2+ ions, which have a bridging function within this disulphide-free protein molecule.

The Role of Calcium in the Isolation of Brush Borders from Epithelial Cells of Rat Small Intestine

1966 ◽  
Vol 1 (4) ◽  
pp. 415-424
Author(s):  
P. F. MILLINGTON ◽  
D. R. CRITCHLEY ◽  
P.W. A. TOVELL
Keyword(s):  
Small Intestine ◽  
Calcium Ions ◽  
Chelating Agent ◽  
Tris Buffer ◽  
Good Separation ◽  
The Third ◽  
Low Concentrations ◽  
Brush Borders ◽  
The Stability

A chelating agent such as EDTA or EGTA used with a dilute TRIS buffer at pH 7.2-7.5 was used in order to effect a good separation of brush borders from the epithelium of the small intestine. A good separation was not obtained in low concentrations of TRIS buffer or saline alone. Brush borders were not obtained when the calcium-chelate complex of EDTA or EGTA was used, and only a partial fractionation was obtained when the magnesium complex of EDTA was tried. The involvement of calcium was further illustrated by adding calcium salts directly to the fractionation medium; separation was prevented when sufficient calcium had been added to saturate the chelating agent. It was found that there was no precise optimum concentration for EDTA but a separation could not be obtained below 2.4 mM/1. The effect of changing the pH of the buffer was also investigated and it was demonstrated that the onset of the ability to release brush borders coincided approximately with the ionization of the third acid radical of the chelating agent. This is in keeping with the suggested hypothesis that EDTA acts by chelating calcium ions. From these and electron-microscope studies it is suggested that the binding of calcium ions is an important factor in the maintenance of the stability of the epithelial cell membrane.

Fluorescence Properties of Hog Kidney Aminoacylase I

1988 ◽  
Vol 43 (9-10) ◽  
pp. 671-678 ◽  
Author(s):  
Thomas vom Bruch ◽  
Klaus-Heinrich Röhm
Keyword(s):  
Ph Dependence ◽  
Fluorescence Emission ◽  
Spectroscopic Properties ◽  
Fluorescence Emission Spectrum ◽  
Native Conformation ◽  
Similar Reduction ◽  
Ans Binding ◽  
Radiationless Energy Transfer ◽  
Tryptophan Residues ◽  
Hog Kidney

Abstract The state of the tryptophan residues of porcine kidney aminoacylase I (EC 3.5.1.14) was investigated by fluorescence spectroscopy and chemical modification. The pH-dependence of the fluorescence emission spectrum of the enzyme indicates that its native conformation prevails between pH 6 and 9.5. Within this range, the ionization of a residue with an apparent pKa of 7.1 quenches the enzyme fluorescence by about 15%. A similar reduction of fluorescence intensity accompanies the inactivation of aminoacylase I by treatment with N-bromosuccinimide in low excess. This suggests that in both cases a single tryptophyl residue out of eight residues per subunit is affected. Quenching by iodide revealed that, in the native conformation of the enzyme, 5-6 tryptophans per subunit are accessible, while 2-3 are buried within the protein. 8-Anilinonaphthalene- L-sulfonate (ANS) is tightly bound to aminoacylase I (1 mol/mol dimer, Kd < 1 μᴍ). ANS binding does not interfere with substrate turnover; the spectroscopic properties of the aminoacylase- ANS complex are consistent with bound ANS being excited by radiationless energy transfer (RET) from buried tryptophyl residues of the enzyme.

Synthesis and Characterization of CdTe/PAA/P-4-VP Quantum Dots

2009 ◽  
Vol 60-61 ◽  
pp. 141-145
Author(s):  
Yu Hua Jiang ◽  
Ji Mei Zhang ◽  
Zhao Dai ◽  
Shi Chao Xu ◽  
Yong Yin ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Fluorescence Emission ◽  
Red Shift ◽  
Carboxyl Groups ◽  
Fluorescence Emission Spectrum ◽  
Characteristic Absorption Peak ◽  
Cdte Qds ◽  
Transmission Electron ◽  
The Stability ◽  
Average Size

In this article, a novel kind of nanomaterials called CdTe/PAA/P-4-VP quantum dots (QDs) was studied. CdTe quantum dots were synthesized by mixing CdCl2 solution containing Mercaptopropionic Acid (MPA) stabilizer at pH 9.1 with NaHTe aqueous solution and refluxing 1 h at 96 °C. Then, CdTe solution obtained was adjusted to pH 7.5 by poly(acrylic acid) (PAA) which could provide many carboxyl groups to CdTe QDs. PAA-modified CdTe (CdTe/PAA)solution was transparent during 2 months and the fluorescence of CdTe/PAA was higher than that of CdTe at the same pH, which indicated that PAA increased the stability and fluorescence intension of CdTe QDs. Then poly(4-vinylpyridine)-coated CdTe/PAA QDs (CdTe/PAA/P-4-VP QDs) were prepared after 4-VP was polymerized on the surface of CdTe/PAA nanoparticles by using N, N'-Methylenebisacrylamide as crosslinker and potassium persulfate as initiator. The parallel experiment in which PAA was replaced by HCl was also arranged and CdTe/P-4-VP QDs was gained. Fluorescence emission spectrum (FE), transmission electron microscope (TEM), and infrared spectrum (IR) were used to characterize CdTe/PAA/P-4-VP and CdTe/P-4-VP QDs. The FE spectrum of CdTe/PAA/P-4-VP QDs had an obviously red shift compared with that of CdTe QDs while the red shift in FE spectrum of CdTe/P-4-VP QDs was not obvious, which indicated that PAA played an important role in the synthesis of P-4-VP-coated CdTe QDs. The fluorescence of CdTe/PAA/P-4-VP QDs was one times higher than that of CdTe QDs, which suggested the advantage of CdTe/PAA/P-4-VP QDs in fluorescence application. TEM photos showed that the average size of CdTe/PAA/P-4-VP QDs was 13.3nm. The IR spectrum of CdTe/PAA/P-4-VP QDs showed that characteristic absorption peak of carboxyl groups from PAA or/and MPA was wide and other absorption peaks of CdTe/PAA/P-4-VP QDs were the same with that of 4-VP which confirmed that P-4-VP coated CdTe QDs.

scholarly journals Structure—function relationship of xylanase: fluorimetric analysis of the tryptophan environment

1996 ◽  
Vol 315 (2) ◽  
pp. 583-587 ◽  
Author(s):  
Kavita R. BANDIVADEKAR ◽  
Vasanti V. DESHPANDE
Keyword(s):  
Structure Function ◽  
Active Site ◽  
Guanidine Hydrochloride ◽  
Fluorescence Emission ◽  
Tryptophan Residue ◽  
Emission Maximum ◽  
Complete Inactivation ◽  
Tryptophan Residues ◽  
Structure Function Relationship ◽  
Function Relationship

Involvement of one out of three tryptophan residues in the active site of the low-molecular-mass xylanase from Chainia has been demonstrated previously [Deshpande, Hinge and Rao (1990) Biochim. Biophys. Acta 1041, 172–177]. The work described here aims at: (i) deducing the structure–function relationship for the tryptophan residue involved at the active site (a) by correlating the effect of N-bromosuccinimide (NBS) on the fluorescence and activity, and (b) by assessing the ability of xylan to protect against decrease in fluorescence versus activity of NBS-treated enzyme; and (ii) probing into the environment of the tryptophan residues by studying the quenching of their fluorescence by various solute quenchers in the presence and absence of guanidine hydrochloride (Gdn.HCl). Complete inactivation of the NBS-treated enzyme occurs well before the loss of fluorescence. Full protection by xylan (0.5%) of the inactivation of enzyme by NBS compared with 30% protection for the decrease in fluorescence confirms the participation of a single tryptophan at the substrate-binding site of the xylanase. The xylanase exhibited a rather low fluorescence emission maximum at 310 nm. There was no shift in the emission maximum on treatment of the enzyme with Gdn.HCl (6.5 M), indicating the rigidity of the microenvironment around tryptophan residues. The quenching studies with acrylamide suggested the occurrence of both collisional as well as static quenching processes. The enzyme retained full activity as well as the characteristic emission maximum at 310 nm in the presence of acrylamide (100 mM), indicating that quenching of fluorescence by acrylamide is a physical process. Acrylamide was more efficient as a quencher than CsCl or KBr. Treatment of the enzyme with Gdn.HCl resulted in an increase in accessibility of the quenchers to the fluorophore as suggested by an increase in the Stern–Volmer quenching constants (Ksv) of the solute quenchers. The analysis of Ksv and V values of KBr and CsCl suggests that the overall tryptophan microenvironment in the xylanase from Chainia is slightly electronegative.

scholarly journals The role of calcium ions in the stability and instability of a thermolysin-like protease

2011 ◽  
Vol 20 (8) ◽  
pp. 1346-1355 ◽  
Author(s):  
V. G. H. Eijsink ◽  
B. W. Matthews ◽  
G. Vriend
Keyword(s):  
Calcium Ions ◽  
Stability And Instability ◽  
The Stability

Mitochondrial matrix calcium ions regulate energy production in myocardium: A cytochemical study

1990 ◽  
Vol 48 (2) ◽  
pp. 166-167
Author(s):  
W.A. Jacob ◽  
R. Hertsens ◽  
A. Van Bogaert ◽  
M. De Smet
Keyword(s):  
Energy Metabolism ◽  
Calcium Ions ◽  
Energy Production ◽  
Regulation Of Respiration ◽  
Free Calcium ◽  
Mitochondrial Matrix ◽  
Cytochemical Study ◽  
Nmr Techniques

In the past most studies of the control of energy metabolism focus on the role of the phosphorylation potential ATP/ADP.Pi on the regulation of respiration. Studies using NMR techniques have demonstrated that the concentrations of these compounds for oxidation phosphorylation do not change appreciably throughout the cardiac cycle and during increases in cardiac work. Hence regulation of energy production by calcium ions, present in the mitochondrial matrix, has been the object of a number of recent studies.Three exclusively intramitochondnal dehydrogenases are key enzymes for the regulation of oxidative metabolism. They are activated by calcium ions in the low micromolar range. Since, however, earlier estimates of the intramitochondnal calcium, based on equilibrium thermodynamic considerations, were in the millimolar range, a physiological correlation was not evident. The introduction of calcium-sensitive probes fura-2 and indo-1 made monitoring of free calcium during changing energy metabolism possible. These studies were performed on isolated mitochondria and extrapolation to the in vivo situation is more or less speculative.

A Statistical Study of Process Variables to Optimize a High Speed Curtain Coater: Part I

TAPPI Journal ◽  
2009 ◽  
Vol 8 (1) ◽  
pp. 20-26 ◽  
Author(s):  
PEEYUSH TRIPATHI ◽  
MARGARET JOYCE ◽  
PAUL D. FLEMING ◽  
MASAHIRO SUGIHARA
Keyword(s):  
Boundary Layer ◽  
Experimental Design ◽  
High Speed ◽  
Statistical Study ◽  
Process Variables ◽  
High Speeds ◽  
The Stability ◽  
Air Removal ◽  
Removal System

Using an experimental design approach, researchers altered process parameters and material prop-erties to stabilize the curtain of a pilot curtain coater at high speeds. Part I of this paper identifies the four significant variables that influence curtain stability. The boundary layer air removal system was critical to the stability of the curtain and base sheet roughness was found to be very important. A shear thinning coating rheology and higher curtain heights improved the curtain stability at high speeds. The sizing of the base sheet affected coverage and cur-tain stability because of its effect on base sheet wettability. The role of surfactant was inconclusive. Part II of this paper will report on further optimization of curtain stability with these four variables using a D-optimal partial-facto-rial design.

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