scholarly journals Further characterization of the covalent linking reaction of α2-macroglobulin

1981 ◽  
Vol 195 (2) ◽  
pp. 453-461 ◽  
Author(s):  
G S Salvesen ◽  
C A Sayers ◽  
A J Barrett
Keyword(s):  
Steric Hindrance ◽  
Peptide Mapping ◽  
Thiol Group ◽  
Molar Ratio ◽  
Reactive Site ◽  
Reactive Group ◽  
Α2 Macroglobulin ◽  
Free Thiol Group ◽  
Covalently Linked

It is shown that non-proteolytic proteins can become covalently linked to alpha 2M (alpha 2-macroglobulin) during its reaction with proteinases, and that this probably occurs by the mechanism that leads to the covalent linking of proteinases described previously [Salvesen & Barrett (1980) Biochem. J. 187, 695-701]. The covalent linking of trypsin was at least partly dependent on the presence of unblocked lysine side chains on the protein. The covalent linking of proteinases was inhibited by nucleophiles of low Mr, and these compounds were themselves linked to alpha 2M in a molar ratio approaching one per quarter subunit. Peptide “mapping” indicated that the site of proteinase-mediated incorporation of the amines was the same as that at which methylamine is incorporated in the absence of a proteinase. The nucleophile-reactive site revealed in alpha 2M after reaction with a proteinase was shown to decay with a t1/2 of 112 s, at pH 7.5. After the reaction with a proteinase or with methylamine, a free thiol group was detectable on each subunit of alpha 2M. We propose that the site for incorporation of methylamine in each subunit is a thiol ester, which in S-alpha 2M (the electrophoretically “slow” form) is sterically shielded from reaction with large nucleophiles, but is revealed as a highly reactive group, free from steric hindrance, after the proteolytic cleavage. We have designated the activated species of the molecule “lpha 2M”.

scholarly journals SYNTHESIS AND CHARACTERIZATION OF HYDROXYPROPYL CELLULOSE-CYSTEAMINE CONJUGATE AS A NOVEL CATIONIC THIOMER WITH LIPOPHILIC PROPERTIES

2019 ◽  
Vol 11 (1) ◽  
pp. 222 ◽  
Author(s):  
Deni Rahmat ◽  
Fikry A. R. Rahman ◽  
Liliek Nurhidayati ◽  
Dian Ratih Laksmitawati
Keyword(s):  
Reductive Amination ◽  
Ph Value ◽  
Thiol Group ◽  
Hydroxypropyl Cellulose ◽  
Scanning Calorimetry ◽  
Swelling Behaviour ◽  
Chemically Modified ◽  
Time Period ◽  
Free Thiol Group

Objective: Thiomers have been known as polymer with mucoadhesive properties. The aim of this study was to synthesize the mucoadhesive potential of hydroxypropyl cellulose-cysteamine conjugate (HPC-cysteamine).Methods: The parent polymer HPC was chemically modified by introducing sulphydryl bearing compound using reductive amination. HPC-cysteamine conjugates were prepared at reaction pH value of 5. The reaction was stabilized by the addition of cyanoborohydride. Afterwards, the conjugate was evaluated for optimum free thiol group, swelling behavior, viscosity and mucoadhesive properties.Results: The conjugates showed maximum thiol incorporation on HPC of 1063.03±64.27 µmol/g. The disulphide groups content was 278.71±32.14 μmol/g. Mucoadhesion studies revealed that mucoadhesion of HPC-cysteamine demonstrated 26 h. The swelling behaviour of HPC-cysteamine tablets increased within the time period of study. The viscosity of HPC-cysteamine was higher than that of unmodified HPC. The thermal profile of HPC-cysteamine and unmodified HPC analyzed by differential scanning calorimetry (DSC) displayed a different enthalpy (ΔH) value.Conclusion: HPC-cysteamine conjugate renders better properties which might be more beneficial for drug delivery system compared to unmodified HPC. 

Post-proline endopeptidase. Interaction of the enzyme with substrates containing disulfide and thioether bond

1986 ◽  
Vol 51 (1) ◽  
pp. 234-240 ◽  
Author(s):  
Karel Hauzer ◽  
Tomislav Barth ◽  
Linda Hauzerová ◽  
Jana Barthová ◽  
Pavel Hrbas ◽  
...  
Keyword(s):  
Arginine Vasopressin ◽  
Thiol Group ◽  
Disulfide Bridge ◽  
Complex Interaction ◽  
Enzyme Inactivation ◽  
Molar Ratio ◽  
Disulfide Exchange ◽  
Neurohypophysial Hormones ◽  
Free Thiol ◽  
Free Thiol Group

The free thiol group of post-proline endopeptidase (EC 3.4.21.26) can interact with the disulfide bridge contained in some of the substrates of this enzyme (neurohypophysial hormones and some of their analogues). The influence of these interactions on the activity of this enzyme was studied using several substances modelling individual types of interactions: thiol-disulfide exchange, catalytic interaction and a complex interaction including the two preceding types. Deamino-1-carba-oxytocin is catalytically hydrolysed in the concentration range up to 10-3mol/l, oxytocin and arginine-vasopressin are catalytically hydrolysed in concentrations of 10-5 to 10-8 mol/l. A reaction leading to inactivation of the enzyme prevails at concentrations of 10-3 to 10-4 mol/l. When inactivated by lower concentrations of arginine-vasopressin (up to a molar ratio of 1 : 1), the enzyme can be reactivated by incubation with dithiothreitol, higher concentrations of arginine-vasopresson cause irreversible enzyme inactivation.

scholarly journals Human cathepsin B1. Inhibition by α2-macroglobulin and other serum proteins

1973 ◽  
Vol 131 (4) ◽  
pp. 823-831 ◽  
Author(s):  
Phyllis M. Starkey ◽  
Alan J. Barrett
Keyword(s):  
Cathepsin B ◽  
Serum Proteins ◽  
Iodoacetic Acid ◽  
Active Enzyme ◽  
Molar Ratio ◽  
High Concentration ◽  
Α2 Macroglobulin ◽  
Normal Human ◽  
Human Cathepsin ◽  
Covalently Linked

1. Normal human serum was found to inhibit human cathepsin B1. 2. The major inhibitor present in serum was purified and identified as α2-macroglobulin. 3. α2-Macroglobulin was found to bind cathepsin B1 in an approximately 1:1 molar ratio. When bound, the enzyme retained about 50% of its proteolytic activity, and up to 80% of its activity against α-N-benzoyl-dl-arginine 2-naphthylamide. 4. Pretreatment of α2-macroglobulin with cathepsin B1 inactivated by exposure to pH8.5 or iodoacetic acid, in large molar excess, did not prevent the subsequent binding of active enzyme. Active enzyme, once bound, was not protected from inhibition by 1-chloro-4-phenyl-3-tosylamido-l-butan-2-one. 5. Cathepsin B1 was also inhibited by human immunoglobulin G, at high concentration. 6. Because it had been suggested that haptoglobin is responsible for the inhibition of ‘cathepsin B’ by serum, a method was devised for the selective removal of haptoglobin from mixtures of serum proteins by adsorption on haemoglobin covalently linked to Sepharose. No evidence was obtained that haptoglobin has any inhibitory activity against the enzyme.

scholarly journals Evolution of α2-macroglobulin. The structure of a protein homologous with human α2-macroglobulin from plaice (Pleuronectes platessa L.) plasma

1982 ◽  
Vol 205 (1) ◽  
pp. 105-115 ◽  
Author(s):  
Phyllis M. Starkey ◽  
Alan J. Barrett
Keyword(s):  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Complement Components ◽  
Pleuronectes Platessa ◽  
Reducing Conditions ◽  
Α2 Macroglobulin ◽  
Mild Reduction ◽  
Covalently Linked

The plaice (Pleuronectes platessa L.) papain-binding protein previously demonstrated to be homologous with human α2-macroglobulin, and designated plaice α2-macroglobulin homologue or αMh, was shown to be a glycoprotein of s20,w 11.86S. In polyacrylamide-gel pore-limit electrophoresis under non-denaturing conditions plaice αMh migrated to the same position as half-molecules of human α2-macroglobulin, and treatment with methylamine or a proteinase caused no change in its electrophoretic properties. Either denaturation in urea (4m) or mild reduction by dithiothreitol (1mm) partially dissociated plaice αMh into half-molecules. Denaturation with reduction further dissociated the protein into quarter-subunits. In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing conditions plaice αMh dissociated into subunits of Mr 105000 (I) and 90000 (II). Approximately equal amounts of each subunit were formed, and peptide ‘mapping’ showed subunits I and II to be distinct polypeptide chains. Under alkaline denaturing conditions, a proportion of the I chains of αMh were cleaved into fragments of Mr about 60000 and 40000. This cleavage was favoured by reducing conditions and prevented by prior inactivation of the αMh with methylamine. [14C]Methylamine allowed to react with αMh became covalently linked to subunit I. These properties suggested the existence of an autolytic site on subunit I analogous to the autolytic site of human α2-macroglobulin. Reaction of αMh with a proteinase resulted in cleavage of a fragment of Mr 10000–15000 from subunit I. A proportion of the proteinase molecules trapped by αMh became covalently linked to the inhibitor. A scheme is proposed for the evolution of human α2-macroglobulin and plaice αMh from a common ancestral protein, which may also have been an ancestor of complement components C3 and C4.

scholarly journals Mouse intracellular immunoglobulin M. Structure and identification of a free thiol group

1978 ◽  
Vol 175 (3) ◽  
pp. 959-967 ◽  
Author(s):  
Neil E. Richardson ◽  
Arnold Feinstein
Keyword(s):  
Plasma Cells ◽  
Peptide Mapping ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Thiol Group ◽  
Immunoglobulin M ◽  
Thiol Groups ◽  
Heavy Chains ◽  
Free Thiol ◽  
Free Thiol Group

Monomeric intracellular mouse immunoglobulin M (hereafter designated IgMs) was purified in milligram quantities from the plasma cells of mouse plasmacytoma MOPC 104E after lysis either in the presence or in the absence of iodoacetate. Peptide ‘mapping’ analysis of the IgMs after partial reduction and carboxy[14C]methylation to label the interchain disulphide bridges showed that the heavy–light bridge and the interheavy bridge present in the Cμ2 region were already formed at lysis. The cysteine residues in the C-terminal region of the heavy chains, which in pentameric IgM form an intersubunit bridge, had free thiol groups at lysis that were reversibly oxidized during isolation in the absence of iodoacetate, probably forming an intrasubunit inter-heavy-chain disulphide bridge. Isoelectric-focusing studies complemented the above findings, showing that all the intracellular IgMs carried free thiol groups that could be carboxymethylated at lysis, and that in non-alkylated preparations these had reversibly oxidized. On the basis of sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis intracellular μ-chains had a consistently lower apparent molecular weight than did secreted μ-chains, and the estimated difference could be accounted for by the known difference in carbohydrate content. We present evidence that in a position homologous to that of a complex oligosaccharide in the Cμ2 region of secreted human μ-chains there is a simple oligosaccharide in intracellular mouse μ-chains that becomes complex on secretion. On the basis of the above findings, we present a model for the mouse intracellular IgM subunit and suggest a mechanism for its assembly into secreted IgM pentamers.

scholarly journals Synthesis, Characterization of Chitosan-Aluminum Oxide Nanocomposite for Green Synthesis of Annulated Imidazopyrazol Thione Derivatives

Polymers ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1160
Author(s):  
Abir S. Abdel-Naby ◽  
Sara Nabil ◽  
Sarah Aldulaijan ◽  
Ibtisam M. Ababutain ◽  
Azzah I. Alghamdi ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Aluminum Oxide ◽  
Reaction Medium ◽  
Thiol Group ◽  
Docking Studies ◽  
Significant Loss ◽  
Gram Negative Bacteria ◽  
Organic Catalyst ◽  
Group 1

Chitosan-aluminum oxide nanocomposite was synthesized, characterized, and used as a green heterogeneous catalyst to synthesize novel imidazopyrazolylthione derivatives. Nanocomposite polymeric material was characterized by EDS-SEM and XRD. The powerful catalytic activity, and its base character of the nanocomposite, was used to synthesize imidazopyrazolylthione (1) in a good yield compared to traditional cyclocondensation synthesis. Using the nanocomposite catalyst, substitution of the thiol group (1) afforded the corresponding thiourea (2) and the corresponding ester (3). The efficiency of the nanocomposite over the traditional base organic catalyst, Et3N and NaOH, makes it an effective, economic, and reproducible nontoxic catalyst. Moreover, the heterogeneous nanocomposite polymeric film was easily isolated from the reaction medium, and recycled up to four times, without a significant loss of its catalytic activity. The newly synthesized derivatives were screened as antibacterial agents and showed high potency. Molecular docking was also performed for a more in-depth investigation. The results of the docking studies have demonstrated that the docked compounds have strong interaction energies with both Gram-positive and Gram-negative bacteria.

scholarly journals 1250. Novel Boronic Acid Transition State Analogs (BATSI) with in vitro inhibitory activity against class A, B and C β-lactamases

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S643-S643
Author(s):  
Maria F Mojica ◽  
Christopher Bethel ◽  
Emilia Caselli ◽  
Magdalena A Taracila ◽  
Fabio Prati ◽  
...  
Keyword(s):  
Active Site ◽  
Inhibitory Activity ◽  
Covalent Bond ◽  
Thiol Group ◽  
Viable Cell ◽  
Free Thiol ◽  
Transition State Analogs ◽  
Cell Counts ◽  
Free Thiol Group

Abstract Background Catalytic mechanisms of serine β-lactamases (SBL; classes A, C and D) and metallo-β-lactamases (MBLs) have directed divergent strategies towards inhibitor design. SBL inhibitors act as high affinity substrates that -as in BATSIs- form a reversible, dative covalent bond with the conserved active site Ser. MBL inhibitors bind the active-site Zn2+ ions and displace the nucleophilic OH-. Herein, we explore the efficacy of a series of BATSI compounds with a free-thiol group at inhibiting both SBL and MBL. Methods Exploratory compounds were synthesized using stereoselective homologation of (+) pinandiol boronates to introduce the amino group on the boron-bearing carbon atom, which was subsequently acylated with mercaptopropanoic acid. Representative SBL (KPC-2, ADC-7, PDC-3 and OXA-23) and MBL (IMP-1, NDM-1 and VIM-2) were purified and used for the kinetic characterization of the BATSIs. In vitro activity was evaluated by a modified time-kill curve assay, using SBL and MBL-producing strains. Results Kinetic assays revealed that IC50 values ranged from 1.3 µM to >100 µM for this series. The best compound, s08033, demonstrated inhibitory activity against KPC-2, VIM-2, ADC-7 and PDC-3, with IC50 in the low μM range. Reduction of at least 1.5 log10-fold of viable cell counts upon exposure to sub-lethal concentrations of antibiotics (AB) + s08033, compared to the cells exposed to AB alone, demonstrated the microbiological activity of this novel compound against SBL- and MBL-producing E. coli (Table 1). Table 1 Conclusion Addition of a free-thiol group to the BATSI scaffold increases the range of these compounds resulting in a broad-spectrum inhibitor toward clinically important carbapenemases and cephalosporinases. Disclosures Robert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)

scholarly journals Synthesis and Characterization of Partially Renewable Oleic Acid-Based Ionomers for Proton Exchange Membranes

Polymers ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 130
Author(s):  
Carlos Corona-García ◽  
Alejandro Onchi ◽  
Arlette A. Santiago ◽  
Araceli Martínez ◽  
Daniella Esperanza Pacheco-Catalán ◽  
...  
Keyword(s):  
Oleic Acid ◽  
Proton Conductivity ◽  
Electrochemical Impedance ◽  
Proton Exchange Membranes ◽  
Proton Exchange ◽  
Molar Ratio ◽  
Aromatic Diamines ◽  
Chemical Structures ◽  
Long Chain

The future availability of synthetic polymers is compromised due to the continuous depletion of fossil reserves; thus, the quest for sustainable and eco-friendly specialty polymers is of the utmost importance to ensure our lifestyle. In this regard, this study reports on the use of oleic acid as a renewable source to develop new ionomers intended for proton exchange membranes. Firstly, the cross-metathesis of oleic acid was conducted to yield a renewable and unsaturated long-chain aliphatic dicarboxylic acid, which was further subjected to polycondensation reactions with two aromatic diamines, 4,4′-(hexafluoroisopropylidene)bis(p-phenyleneoxy)dianiline and 4,4′-diamino-2,2′-stilbenedisulfonic acid, as comonomers for the synthesis of a series of partially renewable aromatic-aliphatic polyamides with an increasing degree of sulfonation (DS). The polymer chemical structures were confirmed by Fourier transform infrared (FTIR) and nuclear magnetic resonance (1H, 13C, and 19F NMR) spectroscopy, which revealed that the DS was effectively tailored by adjusting the feed molar ratio of the diamines. Next, we performed a study involving the ion exchange capacity, the water uptake, and the proton conductivity in membranes prepared from these partially renewable long-chain polyamides, along with a thorough characterization of the thermomechanical and physical properties. The highest value of the proton conductivity determined by electrochemical impedance spectroscopy (EIS) was found to be 1.55 mS cm−1 at 30 °C after activation of the polymer membrane.

scholarly journals Biodiesel Production from Melia azedarach and Ricinus communis Oil by Transesterification Process

Catalysts ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 427 ◽  
Author(s):  
Muhammad Awais ◽  
Sa’ed A Musmar ◽  
Faryal Kabir ◽  
Iram Batool ◽  
Muhammad Asif Rasheed ◽  
...  
Keyword(s):  
Ricinus Communis ◽  
Cost Effective ◽  
Biodiesel Production ◽  
Molar Ratio ◽  
Melia Azedarach ◽  
Renewable Fuel ◽  
Animal Fats ◽  
Edible Crops ◽  
American Society

Biodiesel is a renewable fuel usually produced from vegetable oils and animal fats. This study investigates the extraction of oil and its conversion into biodiesel by base-catalyzed transesterification. Firstly, the effect of various solvents (methanol, n-hexane, chloroform, di-ethyl ether) on extraction of oil from non-edible crops, such as R. communis and M. azedarach, were examined. It was observed that a higher concentration of oil was obtained from R. communis (43.6%) as compared to M. azedarach (35.6%) by using methanol and n-hexane, respectively. The extracted oils were subjected to NaOH (1%) catalyzed transesterification by analyzing the effect of oil/methanol molar ratio (1:4, 1:6, 1:8 and 1:10) and varying temperature (20, 40, 60 and 80 °C) for 2.5 h of reaction time. M. azedarach yielded 88% and R. communis yielded 93% biodiesel in 1:6 and 1:8 molar concentrations at ambient temperature whereas, 60 °C was selected as an optimum temperature, giving 90% (M. azedarach) and 94% (R. communis) biodiesel. The extracted oil and biodiesel were characterized for various parameters and most of the properties fulfilled the American Society for Testing and Materials (ASTM) standard biodiesel. The further characterization of fatty acids was done by Gas Chromatography/Mass Spectrometer (GC/MS) and oleic acid was found to be dominant in M. azedarach (61.5%) and R. communis contained ricinoleic acid (75.53%). Furthermore, the functional groups were analyzed by Fourier Transform Infrared Spectroscopy. The results suggested that both of the oils are easily available and can be used for commercial biodiesel production at a cost-effective scale.

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