Ultraviolet Absorption Spectra of Proteins and Amino Acids

Beaven, G. H.; Holiday, E. R.

doi:10.1016/s0065-3233(08)60022-4

Cited by 1,102 publications

(349 citation statements)

References 102 publications

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“…The rEPOs were stored at ) 40°C as aliquots of solutions in 50 mmol/l sodium chloride and 100 mmol/l sodium phosphate and 0AE02% (w/w) Tween 20 at pH 7AE0. The protein contents of these solutions were determined from their absorbance at 280 nm, assuming a specific absorption coefficient (l/g/cm) at 280 nm of 0AE743 (Davis et al, 1987), after correction for turbidity from their absorption spectra between 320 and 360 nm (Beaven & Holiday, 1952). …”

Section: Erythropoietinsmentioning

confidence: 99%

Relationships between the N‐glycan structures and biological activities of recombinant human erythropoietins produced using different culture conditions and purification procedures

et al. 2003

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Summary. Eight preparations of recombinant human erythropoietin (EPO) with differing isoform compositions were produced by using different culture conditions and purification procedures. The N-glycan structures of these EPOs were analysed using a recently developed profiling procedure and identified using matrix-assisted laser desorption ionization mass spectrometry. The specific activities of each of the EPOs were estimated by in vivo and in vitro mouse bioassays. The eight EPOs were found to differ in their isoform compositions (as judged by isoelectric focusing), their N-glycan profiles, and in their in vivo and in vitro bioactivities. N-glycan analyses identified at least 23 different structures among these EPOs, including bi-, tri-and tetraantennary N-glycans, with or without fucosylation or N-acetyllactosamine extensions, and sialylated to varying degrees. Mass spectrometry also indicated the presence of N-glycans with incomplete outer chains, terminating in N-acetylglucosamine residues, and of molecular masses consistent with phosphorylated or sulphated oligomannoside structures. The tetrasialylated tetra-antennary N-glycan contents of the eight rEPOs were found to be significantly and positively correlated with their specific activities as estimated by mouse in vivo bioassay, and significantly and negatively correlated with their specific activities as estimated by mouse in vitro bioassay. It was concluded that the tetrasialylated tetra-antennary N-glycan content of EPO is a major determinant for its in vivo biological activity in the mouse.

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Section: Erythropoietinsmentioning

confidence: 99%

Relationships between the N‐glycan structures and biological activities of recombinant human erythropoietins produced using different culture conditions and purification procedures

et al. 2003

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“…The concentration of M2 in semipure and purified preparations was estimated from the A280 measurement using the formula of Beaven & Holiday (1952). Concentration estimates took into account the level of impurities as detected by silver staining.…”

Section: Proton Transfer By Influenza Virus M 2 3479mentioning

confidence: 99%

Functional reconstitution in lipid vesicles of influenza virus M2 protein expressed by baculovirus: evidence for proton transfer activity

Schroeder¹,

Ford²,

Wharton³

et al. 1994

Journal of General Virology

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The influenza virus M2 protein was expressed from a recombinant baculovirus in Spodoptera frugiperda Sf9 cells, purified and reconstituted into artificial membrane vesicles. The specific inhibitor amantadine overcame the toxic activity of the protein and boosted the rate of M2 synthesis by a factor of 10, allowing yields of about 1 mg of purified M2 protein per g of Sf9 cells. M2 protein expressed in this system was phosphorylated and palmitoylated and displayed properties similar to the authentic virus protein. Purified wild-type M2 protein and an amantadine-resistant mutant M2 (M26) with a deletion in the trans-membrane domain (amino acids 28 to 31) were incorporated into lipid vesicles, which were loaded with the fluorescent pH indicator pyranine. On imposition of an ionic gradient, M2 caused a decrease in intravesicular pH, which was susceptible to inhibition by 0.1 to 1 gM-rimantadine or N-ethylrimantadine. M26 behaved similarly but exhibited the expected drug resistance. These experiments indicate that isolated M2 functions as an ion channel and demonstrates in vitro M2-mediated proton translocation.

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“…Threonine, serine and tyrosine were corrected for destruction during hydrolysis. Tryptophan content was determined spectrophotometrically [14]. The amount of hexoses in the enzyme preparation was determined with anthrone according to Trevelyan and Harrison [15].…”

Section: Analysis Of Amino Acidsmentioning

confidence: 99%

Beef-Liver Arginase. Isolation and Molecular Properties

Harell

Sokolovsky

1972

Eur J Biochem

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A procedure is described for the purification of arginase from extracts of beef liver. The enzyme which catalyzes the cleavage of L-arginine to ornithine and urea has been purified 630-fold, appeared to be homogeneous in the ultracentrifuge and by disc electrophoresis. The molecular weight determined by sedimentation equilibrium and amino acid analysis is 115000 & 5000. Beef liver arginase is a slightly acidic protein (PI 5.9) and binds 4 atom of Mn2f which are essential to the enzymic activity. The enzyme requires also the presence of manganese ions for stability.Arginase the terminal enzyme of the ureaornithine cycle, catalyzes the cleavage of L-arginine to ornithine and urea [l]. Arginase is present abundantly in mammalian liver [2], but small amounts of arginase are also found in the liver of uricotelic animals, in a variety of plants and in certain bacteria. There are many publications reporting differences in the properties of mammalian arginases 131 (and references cited there). The catalytic activity of arginase from different species and from different tissues has been found to depend greatly on the presence of divalent metal ions, e.g., Mn2+, Co2+, Ni2+ [4-61. Nevertheless, the role of the metal ion in the mode of action of arginase is still unclear. This lack of information prompted us to undertake a study of the molecular properties of beef liver arginase. I n the course of purifying this enzyme using two previously reported procedures [7,8] we were unable to obtain a pure and active arginase preparation suitable for such studies. The work presented here described the development of a new effective method for the purification of beef liver arginase. The enzyme obtained by this procedure has been partially characterized. EXPERIMENTAL PROCEDURE MATERIALSL-Arginine (free base) and L-ornithine-HC1 were obtained from Ajinomoto (Japan). Urea was obtained from Fisher, DEAE-cellulose (DE-52) from Whatman, SE-Sephadex C-50 from Pharmacia, Biogel P-150 (100-200mesh) and P-4 (100 to 200mesh) from Bio-Rad, MnC1,.4H,O and diacetylmonoxime (analytical grade) from Merck, arsenic acid (syrupy) from BDH. Beef livers were Enzyme. Arginase (EC 3.5.3.1). obtained a t the slaughter house, and were chilled immediately in dry ice. The livers were either immediately worked up or frozen at -20 "C. METHODS

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Ultraviolet Absorption Spectra of Proteins and Amino Acids

Cited by 1,102 publications

References 102 publications

Relationships between the N‐glycan structures and biological activities of recombinant human erythropoietins produced using different culture conditions and purification procedures

Relationships between the N‐glycan structures and biological activities of recombinant human erythropoietins produced using different culture conditions and purification procedures

Functional reconstitution in lipid vesicles of influenza virus M2 protein expressed by baculovirus: evidence for proton transfer activity

Beef-Liver Arginase. Isolation and Molecular Properties

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