scholarly journals Structural and Chemical Characterization of Silica Spheres before and after Modification by Silanization for Trypsin Immobilization

2017 ◽  
Vol 2017 ◽  
pp. 1-10
Author(s):  
Eduardo F. Barbosa ◽  
Luciano P. Silva
Keyword(s):  
High Performance ◽  
Morphological Changes ◽  
Chemical Characterization ◽  
Reversed Phase ◽  
Silica Particles ◽  
Selective Hydrolysis ◽  
Silica Surfaces ◽  
Immobilized Trypsin ◽  
Before And After ◽  
Silanized Silica

In the last decades, silica particles of a variety of sizes and shapes have been characterized and chemically modified for several applications, from chromatographic separation to dental supplies. The present study proposes the use of aminopropyl triethoxysilane (APTS) silanized silica particles to immobilize the proteolytic enzyme trypsin for the development of a bioreactor. The major advantage of the process is that it enables the polypeptides hydrolysis interruption simply by removing the silica particles from the reaction bottle. Silanized silica surfaces showed significant morphological changes at micro- and nanoscale level. Chemical characterization showed changes in elemental composition, chemical environment, and thermal degradation. Their application as supports for trypsin immobilization showed high immobilization efficiency at reduced immobilization times, combined with more acidic conditions. Indirect immobilization quantification by reversed-phase ultrafast high performance liquid chromatography proved to be a suitable approach due to its high linearity and sensitivity. Immobilized trypsin activities on nonmodified and silanized silica showed promising features (e.g., selective hydrolysis) for applications in proteins/peptides primary structure elucidation for proteomics. Silanized silica system produced some preferential targeting peptides, probably due to the hydrophobicity of the nanoenvironment conditioned by silanization.

Fertilization in the sea

2002 ◽  
Vol 205 (10) ◽  
pp. 1439-1450 ◽  
Author(s):  
Jeffrey A. Riffell ◽  
Patrick J. Krug ◽  
Richard K. Zimmer
Keyword(s):  
High Performance ◽  
Sea Water ◽  
Chemical Characterization ◽  
Fertilization Success ◽  
Reversed Phase ◽  
Size Exclusion ◽  
Signal Molecules ◽  
Resonance Spectroscopy ◽  
Significant Activity ◽  
Red Abalone

SUMMARYChemical communication between sperm and egg is a key factor mediating sexual reproduction. Dissolved signal molecules that cause sperm to orient and accelerate towards an egg could play pivotal roles in fertilization success,but such compounds are largely undescribed. This investigation considered the behavioral responses of red abalone (Haliotis rufescens) sperm to soluble factors released into sea water by conspecific eggs. Sperm in proximity to individual live eggs swam significantly faster and oriented towards the egg surface. Bioassay-guided fractionation was employed to isolate the chemoattractant, yielding a single pure, fully active compound after reversed-phase and size-exclusion high-performance liquid chromatography. Chemical characterization by nuclear magnetic resonance spectroscopy indicated that the free amino acid L-tryptophan was the natural sperm attractant in H. rufescens.Eggs released L-tryptophan at concentrations that triggered both activation and chemotaxis in sperm, exhibiting significant activity at levels as low as 10-8 mol l-1. The D-isomer of tryptophan was inactive,showing that the sperm response was stereospecific. Serotonin, a potent neuromodulator and tryptophan metabolite, had no effect on sperm swim speeds or on orientation. In experimental treatments involving an elevated, uniform concentration of tryptophan (10-7 mol l-1) or the addition of tryptophanase, an enzyme that selectively digests tryptophan,sperm failed to navigate towards live eggs. A natural gradient of L-tryptophan was therefore necessary and sufficient to promote recruitment of sperm to the surface of eggs in red abalone.

Chymotrypsin selectively digests β-lactoglobulin in whey protein isolate away from enzyme optimal conditions: Potential for native α-lactalbumin purification

2012 ◽  
Vol 80 (1) ◽  
pp. 14-20 ◽  
Author(s):  
Katarina Lisak ◽  
Jose Toro-Sierra ◽  
Ulrich Kulozik ◽  
Rajka Božanić ◽  
Seronei Chelulei Cheison
Keyword(s):  
Whey Protein ◽  
High Performance ◽  
Enzyme Reaction ◽  
Whey Protein Isolate ◽  
Reversed Phase ◽  
Protein Isolate ◽  
Operating Conditions ◽  
Selective Hydrolysis ◽  
Experimental Conditions ◽  
Β Lactoglobulin

The present study examines the resistance of the α-lactalbumin to α-chymotrypsin (EC 3.4.21.1) digestion under various experimental conditions. Whey protein isolate (WPI) was hydrolysed using randomised hydrolysis conditions (5 and 10% of WPI; pH 7·0, 7·8 and 8·5; temperature 25, 37 and 50 °C; enzyme-to-substrate ratio, E/S, of 0·1%, 0·5 and 1%). Reversed-phase high performance liquid chromatography (RP-HPLC) was used to analyse residual proteins. Heat, pH adjustment and two inhibitors (Bowman–Birk inhibitor and trypsin inhibitor from chicken egg white) were used to stop the enzyme reaction. While operating outside of the enzyme optimum it was observed that at pH 8·5 selective hydrolysis of β-lactoglobulin was improved because of a dimer-to-monomer transition while α-la remained relatively resistant. The best conditions for the recovery of native and pure α-la were at 25 °C, pH 8·5, 1% E/S ratio, 5% WPI (w/v) while the enzyme was inhibited using Bowman–Birk inhibitor with around 81% of original α-la in WPI was recovered with no more β-lg. Operating conditions for hydrolysis away from the chymotrypsin optimum conditions offers a great potential for selective WPI hydrolysis, and removal, of β-lg with production of whey protein concentrates containing low or no β-lg and pure native α-la. This method also offers the possibility for production of β-lg-depleted milk products for sensitive populations.

Comparison of selective hydrolysis of α-lactalbumin by acid Protease A and Protease M as alternative to pepsin: potential for β-lactoglobulin purification in whey proteins

2019 ◽  
Vol 86 (1) ◽  
pp. 114-119
Author(s):  
Katarina Lisak Jakopović ◽  
Seronei Chelulei Cheison ◽  
Ulrich Kulozik ◽  
Rajka Božanić
Keyword(s):  
High Performance ◽  
Whey Proteins ◽  
Reversed Phase ◽  
Protein Isolate ◽  
Acid Protease ◽  
Selective Hydrolysis ◽  
Hydrolysis Conditions ◽  
Β Lactoglobulin ◽  
Hydrolysis Of ◽  
Enzyme Selectivity

AbstractThe experiments reported in this research paper examine the potential of digestion using acidic enzymes Protease A and Protease M to selectively hydrolyse α-lactalbumin (α-La) whilst leaving β-lactoglobulin (β-Lg) relatively intact. Both enzymes were compared with pepsin hydrolysis since its selectivity to different whey proteins is known. Analysis of the hydrolysis environment showed that the pH and temperature play a significant role in determining the best conditions for achievement of hydrolysis, irrespective of which enzyme was used. Whey protein isolate (WPI) was hydrolysed using pepsin, Acid Protease A and Protease M by randomized hydrolysis conditions. Reversed-phase high performance liquid chromatography was used to analyse residual proteins. Regarding enzyme selectivity under various milieu conditions, all three enzymes showed similarities in the reaction progress and their potential for β-Lg isolation.

Direct, Simplified, and Sensitive Assay of Angiotensin II in Plasma Extracts Performed with a High-Affinity Monoclonal Antibody

1992 ◽  
Vol 38 (10) ◽  
pp. 1963-1967 ◽  
Author(s):  
D Simon ◽  
B Romestand ◽  
H Huang ◽  
G Badouaille ◽  
J A Fehrentz ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Human Plasma ◽  
High Performance ◽  
Solid Phase ◽  
High Performance Liquid Chromatographic ◽  
Reversed Phase ◽  
Cross Reactivity ◽  
Ang Ii ◽  
High Affinity ◽  
Before And After

Abstract A very simple, fast, and sensitive RIA of angiotensin (Ang) II has been developed, based on a monoclonal antibody with high affinity and specificity, making possible the direct measurement of circulating Ang II in human plasma after solid-phase extraction. The purified monoclonal antibody 4D8 has an association constant of 1.3 x 10(11) L/mol with Ang II and a cross-reactivity of < 1% for Ang I. The assay can detect as little as 0.8 fmol of Ang II in 2 mL of plasma and is not influenced by the presence of Ang I. Analytical recoveries between 112% and 116% were obtained for Ang II added to human plasma at physiological concentrations. Comparison of the RIA with a reversed-phase, high-performance liquid chromatographic method followed by RIA to measure Ang II in human plasma samples from normal and hypertensive subjects--and from normotensive subjects before and after an acute inhibition of angiotensin-converting enzyme with captopril (50 mg)--showed a high degree of correlation (r2 = 0.93) between the two methods.

Serum Steroid Hormonal Profiles by Reversed-Phase Liquid Chromatography in Patients with 17-Hydroxylase Deficiency and in an Affected Family

1992 ◽  
Vol 38 (1) ◽  
pp. 76-82 ◽  
Author(s):  
Jl-Qing Wei ◽  
Ji-Lu Wei ◽  
Claudio Lucarelli ◽  
Xian-Teng Zhou ◽  
De-Quan Wang ◽  
...  
Keyword(s):  
High Performance ◽  
Reversed Phase ◽  
Primary Amenorrhea ◽  
Hydroxylase Deficiency ◽  
Normotensive Patient ◽  
Normal Ranges ◽  
Sigma B ◽  
Before And After ◽  
21 Hydroxylase Deficiency ◽  
Corticotropin Stimulation

Abstract We used an optimized isocratic reversed-phase high-performance liquid-chromatographic procedure to separate and measure 12 steroid hormones, and studied the steroid hormone profiles in sera from three patients with 17-hydroxylase deficiency (17-OHD). Two of the patients were sisters, one of whom (II-3), expressing normotension and primary amenorrhea, was diagnosed on the basis of chromatographic data and followed up for seven years. The untreated patients had obvious abnormalities on chromatograms of serum extracts, characterized by markedly increased corticosterone (B) and decreased or undetectable cortisol (F) and cortisone (E). The concentration of 11-deoxycorticosterone was much greater in the patient with classical symptoms than in the normotensive patient. In all three patients, concentrations of aldosterone were within the normal range, but concentrations of progesterone were much lower than in the patients with 21-hydroxylase deficiency. We evaluated the responses to corticotropin and dexamethasone. HPLC evaluation of the serum steroid profiles before and after corticotropin stimulation in the affected family showed that in the parents and one other sibling, concentrations of F before and after stimulation were within the normal ranges. The sums of the ratio of B to F before and the ratio of B to F after corticotropin stimulation (sigma B/F) in the parents and the other sibling were 0.292, 0.496, and 0.614, respectively, all much higher than the normal value (mean +/- SD: 0.164 +/- 0.038). Thus the sigma B/F value may be a hormonal marker of heterozygotes carrying this defect.

scholarly journals Pressurized planar electrochromatography of DNS amino acids derivatives in silica gel and silanized silica gel systems with formic acid addition to the water mobile phase

2021 ◽  
Author(s):  
Adam Chomicki ◽  
Tadeusz H. Dzido
Keyword(s):  
Amino Acids ◽  
Silica Gel ◽  
High Performance ◽  
Reversed Phase ◽  
Mobile Phases ◽  
Phase Systems ◽  
Silanized Silica ◽  
Layer Chromatography ◽  
Derivatives Of ◽  
Electrophoretic Effect

AbstractPressurized planar electrochromatography (PPEC) of dansyl (DNS) derivatives of amino acids in normal- and reversed-phase systems is presented. The results have been obtained for mobile phases with different acetonitrile (ACN) concentrations (0–85%). The data obtained show differences in separation selectivity between high-performance thin-layer chromatography (HPTLC) and PPEC systems. These differences originate from the electrophoretic effect which is involved in the PPEC system, contrary to the HPTLC one.

scholarly journals Identification of Food-Derived Bioactive Peptides in Blood and Other Biological Samples

2008 ◽  
Vol 91 (4) ◽  
pp. 995-1001 ◽  
Author(s):  
Kenji Sato ◽  
Koji Iwai ◽  
Misako Aito-Inoue
Keyword(s):  
High Performance ◽  
Solid Phase ◽  
Reversed Phase ◽  
Size Exclusion ◽  
Phenyl Isothiocyanate ◽  
Rp Hplc ◽  
Specificity And Sensitivity ◽  
Spin Column ◽  
Before And After ◽  
Hydrophilic Peptide

Abstract Recent studies have demonstrated the presence of food-derived peptides in human blood after ingestion of enzymatic hydrolysates of food proteins, while most peptides in food are degraded into amino acids during digestion and absorption. To capture and clarify the food-derived peptides in blood, solid-phase extraction (SPE) using a mini-spin column packed with a strong cation exchanger was developed. This technique allows the use of a nonvolatile acid such as trichloroacetic acid, a strong protein denaturant, for the deproteinizing procedure. To improve resolution of hydrophilic peptide and increase specificity and sensitivity in the detection of peptide by reversed-phase high-performance liquid chromatography (RP-HPLC) after subfractionation by size-exclusion chromatography (SEC), peptides are derivatized with phenyl isothiocyanate. The resultant phenyl thiocarbamyl (PTC)-peptides can be resolved with high resolution and sensitivity by RP-HPLC. By comparing chromatograms of PTC derivatives from blood before and after ingestion of a peptide sample, food-derived peptide can be detected. The isolated PTC-peptide can be applied to a peptide sequencer based on the Edman degradation reaction.

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