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Proteinase K
In molecular biology, Proteinase K (, ''protease K'', ''endopeptidase K'', ''Tritirachium alkaline proteinase'', ''Tritirachium album serine proteinase'', ''Tritirachium album proteinase K'') is a broad-spectrum serine protease. The enzyme was discovered in 1974 in extracts of the fungus '' Parengyodontium album'' (formerly '' Engyodontium album or Tritirachium album''). Proteinase K is able to digest hair (keratin), hence, the name "Proteinase K". The predominant site of cleavage is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha amino groups. It is commonly used for its broad specificity. This enzyme belongs to Peptidase family S8 (subtilisin). The molecular weight of Proteinase K is 28,900 daltons (28.9 kDa). Enzyme activity Activated by calcium, the enzyme digests proteins preferentially after hydrophobic amino acids (aliphatic, aromatic and other hydrophobic amino acids). Although calcium ions do not affect the e ...
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Molecular Biology
Molecular biology is a branch of biology that seeks to understand the molecule, molecular basis of biological activity in and between Cell (biology), cells, including biomolecule, biomolecular synthesis, modification, mechanisms, and interactions. Though cells and other microscopic structures had been observed in living organisms as early as the 18th century, a detailed understanding of the mechanisms and interactions governing their behavior did not emerge until the 20th century, when technologies used in physics and chemistry had advanced sufficiently to permit their application in the biological sciences. The term 'molecular biology' was first used in 1945 by the English physicist William Astbury, who described it as an approach focused on discerning the underpinnings of biological phenomena—i.e. uncovering the physical and chemical structures and properties of biological molecules, as well as their interactions with other molecules and how these interactions explain observ ...
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Guanidinium Chloride
Guanidinium chloride or guanidine hydrochloride, usually abbreviated GdmCl and sometimes GdnHCl or GuHCl, is the hydrochloride salt of guanidine. Structure Guanidinium chloride on a weighing boat Guanidinium chloride crystallizes in orthorhombic space group ''Pbca''. The crystal structure consists of a network of guanidinium cations and chloride anions linked by N–H···Cl hydrogen bonds. Acidity Guanidinium chloride is a weak acid with a pKa of 13.6. The reason that it is such a weak acid is the complete delocalization of the positive charge through three nitrogen atoms (plus a little bit of positive charge on carbon). However, some stronger bases can deprotonate it, such as sodium hydroxide: : The equilibrium is not complete because the acidity difference between guanidinium and water is not large. The approximate pKa values: 13.6 vs 15.7. Complete deprotonation should be done with extremely strong bases, such as lithium diisopropylamide. : Use in protein dena ...
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Nuclease
In biochemistry, a nuclease (also archaically known as nucleodepolymerase or polynucleotidase) is an enzyme capable of cleaving the phosphodiester bonds that link nucleotides together to form nucleic acids. Nucleases variously affect single and double stranded breaks in their target molecules. In living organisms, they are essential machinery for many aspects of DNA repair. Defects in certain nucleases can cause genetic instability or immunodeficiency. Nucleases are also extensively used in molecular cloning. There are two primary classifications based on the locus of activity. Exonucleases digest nucleic acids from the ends. Endonucleases act on regions in the ''middle'' of target molecules. They are further subcategorized as deoxyribonucleases and ribonucleases. The former acts on DNA, the latter on RNA. History In the late 1960s, scientists Stuart Linn and Werner Arber isolated examples of the two types of enzymes responsible for phage growth restriction in Escherichi ...
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Nucleic Acid
Nucleic acids are large biomolecules that are crucial in all cells and viruses. They are composed of nucleotides, which are the monomer components: a pentose, 5-carbon sugar, a phosphate group and a nitrogenous base. The two main classes of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). If the sugar is ribose, the polymer is RNA; if the sugar is deoxyribose, a variant of ribose, the polymer is DNA. Nucleic acids are chemical compounds that are found in nature. They carry information in cells and make up genetic material. These acids are very common in all living things, where they create, encode, and store information in every living cell of every outline of life forms, life-form on Earth. In turn, they send and express that information inside and outside the cell nucleus. From the inner workings of the cell to the young of a living thing, they contain and provide information via the nucleic acid sequence. This gives the RNA and DNA their unmistakable 'la ...
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Iodoacetic Acid
Iodoacetic acid is an organic compound with the chemical formula . It is a derivative of acetic acid. It is a toxic compound, because, like many alkyl halides, it is an alkylating agent. It reacts with cysteine residues in proteins. It is often used to modify groups to prevent the re-formation of disulfide bonds after the reduction of cystine residues to cysteine during protein sequencing. In 1929, Dr. Einar Lundsgaard (1899-1968) discovered that muscle poisoned in vitro with iodoacetic acid is unable to produce lactate as glycolysis from muscle glycogen is blocked, causing the muscle to result in an electrically silent contracture. It was remembering this discovery, that lead Dr. Brian McArdle in 1951, to speculate that one of his patients that had electromyographically silent muscle contractures brought on by high-intensity aerobic activity and anaerobic activity must have a defective muscle glycogen mechanism. Peptidase inhibitor Iodoacetate is an irreversible inhibitor ...
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Citrate
Citric acid is an organic compound with the formula . It is a colorless weak organic acid. It occurs naturally in citrus fruits. In biochemistry Biochemistry, or biological chemistry, is the study of chemical processes within and relating to living organisms. A sub-discipline of both chemistry and biology, biochemistry may be divided into three fields: structural biology, enzymology, a ..., it is an intermediate in the citric acid cycle, which occurs in the metabolism of all aerobic organisms. More than two million tons of citric acid Commodity chemicals, are manufactured every year. It is used widely as acidifier, flavoring, preservative, and chelating agent. A citrate is a derivative of citric acid; that is, the salt (chemistry), salts, esters, and the polyatomic ion, polyatomic anion found in solutions and salts of citric acid. An example of the former, a salt is trisodium citrate; an ester is triethyl citrate. When citrate anion, trianion is part of a salt, the for ...
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Polysorbate 20
Polysorbate 20 (common commercial brand names include Kolliphor PS 20, Scattics, Alkest TW 20, Tween 20, and Kotilen-20) is a polysorbate-type nonionic surfactant formed by the ethoxylation of sorbitan monolaurate. Its stability and relative nontoxicity allows it to be used as a detergent and emulsifier in a number of domestic, scientific, and pharmacological applications. As the name implies, the ethoxylation process leaves the molecule with 20 repeat units of polyethylene glycol; in practice these are distributed across 4 different chains, leading to a commercial product containing a range of chemical species. Food applications Polysorbate 20 is used as a wetting agent in flavored mouth drops such as Ice Drops, helping to provide a spreading feeling to other ingredients like SD alcohol and mint flavor. The World Health Organization has suggested acceptable daily intake limits of 0–25 mg of polyoxyethylene sorbitan esters per kg body weight. Biotechnical applicatio ...
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Triton X-100
Triton X-100 (''n'') is a nonionic surfactant that has a hydrophilic polyethylene oxide chain (on average it has 9.5 ethylene oxide units) and an aromatic hydrocarbon lipophilic or hydrophobic group. The hydrocarbon group is a 4-( 1,1,3,3-tetramethylbutyl)- phenyl group. Triton X-100 is closely related to IGEPAL CA-630, which might differ from it mainly in having slightly shorter ethylene oxide chains. As a result, Triton X-100 is slightly more hydrophilic than Igepal CA-630 thus these two detergents may not be considered functionally interchangeable for most applications. Triton X-100 was originally a registered trademark of Rohm and Haas Co. It was subsequently purchased by Union Carbide and then acquired by Dow Chemical Company upon the acquisition of Union Carbide. Soon afterward (in 2009), Dow also acquired Rohm & Haas Co. Physical properties Undiluted Triton X-100 is a clear viscous fluid (less viscous than undiluted glycerol). Undiluted Triton X-100 has a viscosity ...
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Sodium Lauroyl Sarcosinate
Sodium lauroyl sarcosinate ( INCI), also known as sarcosyl, is an anionic surfactant derived from sarcosine used as a foaming and cleansing agent in shampoo, shaving foam, toothpaste, and foam wash products. This surfactant is amphiphilic due to the hydrophobic 12-carbon chain ( lauroyl) and the hydrophilic carboxylate. Since the nitrogen atom is in an amide linkage, the nitrogen is not pH active and is neutrally charged in all aqueous solutions regardless of pH. The carboxylate has a pKa of about 3.6 and is therefore negatively charged in solutions of pH greater than about 5.5. pH-sensitive vesicles can be prepared using this surfactant with another cationic or water-insoluble amphiphiles such as 1-decanol. Addition of a mixture of equal parts of sodium lauroyl sarcosinate and the non-ionic surfactant sorbitan monolaurate (S20) to a buffered water:ethanol solution led to the formation of micelle-like aggregates, even though neither surfactant formed micelles when presen ...
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Diisopropyl Fluorophosphate
Diisopropyl fluorophosphate (DFP) or Isoflurophate is an oily, colorless liquid with the chemical formula C6H14FO3P. It is used in medicine and as an organophosphorus insecticide. It is stable, but undergoes hydrolysis when subjected to moisture. Uses in medicine Diisopropyl fluorophosphate is a parasympathomimetic drug irreversible anti-cholinesterase and has been used in ophthalmology as a miotic agent in treatment of chronic glaucoma, as a miotic in veterinary medicine, and as an experimental agent in neuroscience because of its acetylcholinesterase inhibitory properties and ability to induce delayed peripheral neuropathy. Uses as toxin The marked toxicity of esters of monofluorophosphoric acid was discovered in 1932, when Willy Lange and his PhD student Gerda von Krueger prepared the methyl, ethyl, n-propyl, and n-butyl esters and incidentally experienced their toxic effects. Another homologue of this series of esters, Diisopropyl fluorophosphate, was developed by B ...
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PMSF
In biochemistry, phenylmethylsulfonyl fluoride (PMSF) is a serine protease inhibitor (serine hydrolase inactivator) commonly used in the preparation of cell lysates. PMSF does not inactivate all serine proteases. The effective concentration of PMSF is between 0.1 - 1 mM. The half-life is short in aqueous solutions (110 min at pH 7, 55 min at pH 7.5, and 35 min at pH 8, all at 25 °C). At 4˚C, pH 8, PMSF is almost completely degraded after 1 day. Stock solutions are usually made up in anhydrous ethanol, isopropanol, or corn oil and diluted immediately before use. PMSF reacts specifically with the active site serine residue in serine hydrolases. It does not bind to any other serine residues in the protein. This is a result of the hyperactivity of that serine residue caused by the specific environmental conditions in the enzyme's active site (catalytic triad). Because PMSF bonds covalently to the enzyme, the complex can be viewed by X-ray crystallography; it can therefore be u ...
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AEBSF
AEBSF or 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride is a water-soluble, irreversible serine protease inhibitor with a molecular weight of 239.5 Da. It inhibits proteases like chymotrypsin, kallikrein, plasmin, thrombin, and trypsin. The specificity is similar to the inhibitor PMSF, nevertheless AEBSF is more stable at low pH values. Typical usage is 0.1 - 1.0 mM. AEBSF (marketed as Pefabloc SC from the company Pentapharm) was first reported for use in biochemistry in 1993, and came into common use for the inhibition serine proteases and of non-protease enzymes such as acetylhydrolases in the mid 1990s. Mechanism of action AEBSF is targeted to covalently modify the hydroxyl of serine residues, where it causes an additional 183.0354 Da to be added to each modified residue, but other off-target residues such as tyrosine, lysine, histidine, and the protein N-terminal amino group, have also been reported. Due to the substantial frequency of modification of these off ...
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