[10] Protein fractionation on the basis of solubility in aqueous solutions of salts and organic solvents

Green, Arda Alden; Hughes, Walter L.

doi:10.1016/0076-6879(55)01014-8

Cited by 380 publications

(165 citation statements)

References 23 publications

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“…For the initial purification step, the crude extract containing the enzyme was fractionated using the increasing concentrations of ammonium sulphate as described by Green and Hughens (1955). Fractions named AS1, AS2, AS3, AS4 and AS5 were precipitated with 0-20, 20-40, 40-60, 60-80 and 80-100% ammonium sulphate, respectively at 4ºC.…”

Section: Partial Purification Of Milk Clotting Proteasementioning

confidence: 99%

Jacaratia corumbensis O. Kuntze a new vegetable source for milk-clotting enzymes

Duarte

Moreira

et al. 2009

Braz. arch. biol. technol.

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The partial characterization and purification of milk clotting enzyme obtained from the (root latex) of Jacaratia corumbensis O. kuntze was studied, by fractional precipitation with ammonium sulphate and ion exchange chromatography. The ammonium sulphate precipitate showed five fractions and among the fractions obtained, the 40-60% fraction (AS3) showed the highest milk clotting activity with a purification factor of 1.2 fold in relation to the crude extract. This fraction when applied on Mono Q column yielded two protein peaks (p1 and p2), but p1 pool showed the best milk-clotting activity. The optimal pH for the crude and partially purified extract was 6.5 and 7.0, respectively. The maximum milk-clotting activity was at 55ºC for the both crude and partially purified extracts. The enzyme was inhibited by iodoacetic acid which suggested that this enzyme was a cysteine protease, with molecular weight of 33 kDa.

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Section: Partial Purification Of Milk Clotting Proteasementioning

confidence: 99%

Jacaratia corumbensis O. Kuntze a new vegetable source for milk-clotting enzymes

Duarte

Moreira

et al. 2009

Braz. arch. biol. technol.

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“…In this report, the extraction buffer contains the chemicals indicated within the parentheses, but the salt concentration is varied as indicated for different experiments. The solution was stirred gently at room temperature, cleared by centrifugation at 13,000 ϫ g for 30 min at 4°C, and subjected to ammonium sulfate precipitation (15). Ammonium sulfate was added to 70% saturation.…”

Section: Purification Of Peanut Allergen Ara Hmentioning

confidence: 99%

Structure of the Major Peanut Allergen Ara h 1 May Protect IgE-Binding Epitopes from Degradation

Maleki

Kopper²,

Shin³

et al. 2000

The Journal of Immunology

240

210

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In the past decade, there has been an increase in allergic reactions to peanut proteins, sometimes resulting in fatal anaphylaxis. The development of improved methods for diagnosis and treatment of peanut allergies requires a better understanding of the structure of the allergens. Ara h 1, a major peanut allergen belonging to the vicilin family of seed storage proteins, is recognized by serum IgE from >90% of peanut-allergic patients. In this communication, Ara h 1 was shown to form a highly stable homotrimer. Hydrophobic interactions were determined to be the main molecular force holding monomers together. A molecular model of the Ara h 1 trimer was constructed to view the stabilizing hydrophobic residues in the three dimensional structure. Hydrophobic amino acids that contribute to trimer formation are at the distal ends of the three dimensional structure where monomer-monomer contacts occur. Coincidentally, the majority of the IgE-binding epitopes are also located in this region, suggesting that they may be protected from digestion by the monomer-monomer contacts. On incubation of Ara h 1 with digestive enzymes, various protease-resistant fragments containing IgE-binding sites were identified. The highly stable nature of the Ara h 1 trimer, the presence of digestion resistant fragments, and the strategic location of the IgE-binding epitopes indicate that the quaternary structure of a protein may play a significant role in overall allergenicity.

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“…The enzyme was prepared by a modification of the method of Louis & Fitt (1971b). All operations were performed at 0-5°C and (NH4)2SO4 saturation was calculated by the procedure of Green & Hughes (1955).…”

Section: Methodsmentioning

confidence: 99%

Purification and properties of the ribonucleic acid-dependent ribonucleic acid polymerase from Halobacterium cutirubrum

Louis

Fitt

1972

Biochemical Journal

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1. The RNA-dependent RNA polymerase from Halobacterium cutirubrum was purified to electrophoretic homogeneity. 2. It requires a single-stranded molecule of RNA or polyribonucleotide as template. 3. Nearest-neighbour analyses of the products formed on random poly(A,U) or alternating poly(A-U) templates and base analysis of the product of synthesis directed by wheat-germ RNA prove that the template is copied accurately. 4. The enzyme initiates new chains with purine ribonucleoside triphosphates. 5. Sucrose-density-gradient analysis of the product indicates that it has a size distribution similar to that of the template. 6. Preliminary amino acid analysis of the RNA-dependent polymerase shows that it contains much less serine than either of the subunits of H. cutirubrum DNA-dependent RNA polymerase. 7. The RNA-dependent enzyme is unable to substitute for either subunit of the DNA-dependent polymerase, and both the latter are devoid of RNA-dependent activity.

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[10] Protein fractionation on the basis of solubility in aqueous solutions of salts and organic solvents

Cited by 380 publications

References 23 publications

Jacaratia corumbensis O. Kuntze a new vegetable source for milk-clotting enzymes

Jacaratia corumbensis O. Kuntze a new vegetable source for milk-clotting enzymes

Structure of the Major Peanut Allergen Ara h 1 May Protect IgE-Binding Epitopes from Degradation

Purification and properties of the ribonucleic acid-dependent ribonucleic acid polymerase from Halobacterium cutirubrum

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