Protein quantitation at the picomole level: An o-phthaldialdehyde-preTSK column-derivatization assay

Fried, Victor A.; Ando, Michael E.; BEll, A. J.

doi:10.1016/0003-2697(85)90426-9

Cited by 21 publications

(1 citation statement)

References 5 publications

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“…To determine if altered glycosylation of the Chick/Penn HA was sufficient to explain the differences between the avirulent and virulent strains, we (15) or by fluorescent protein assay with bovine serum albumin as standard (16 (18). The phenylthiohydantoin derivatives of aspartic and glutamic residues were identified directly as their methyl esters.…”

mentioning

confidence: 99%

Glycosylation affects cleavage of an H5N2 influenza virus hemagglutinin and regulates virulence.

Deshpande¹,

Fried²,

Ando³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

197

132

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Based on nucleotide sequence analysis of the hemagglutinin (HA) gene from the virulent and avirulent A/chicken/Pennsylvania/83 influeza viruses, it was previously postulated that acquisition of virulence was associated with a point mutation that resulted in loss of a glycosylation site. Since there are two potential glycosylation sites in this region of the HA molecule and since all Asn-Xaa-Thr/Ser sequences in the HAs of different strains are not necessarily glycosylated, the question remained open as to whether either one of these sites was glycosylated. We now provide direct evidence that a site-specific glycosylation affects cleavage of the influenza virus HA and thus virulence. We have identified the glycosylation sites on the HAl subunit from the virulent and avirulent strains by direct structural analysis of the isolated proteins. Our results show that the only difference in glycosylation between the HAls of the virulent and avirulent strains is the lack of an asparagine-linked carbohydrate on the virulent HAl polypeptide at residue 11. Further, we show that the HAls of both the avirulent and virulent viruses are not glycosylated at one potential site, while all other sites contain carbohydrate. Amino acid sequence analysis of the HAl of an avirulent revertant of the virulent strain confirmed these findings.In April 1983, an avirulent (avir) influenza virus, A/chicken/Pennsylvania/1/83 (Chick/Penn) (HSN2), appeared in chickens of eastern Pennsylvania (1). By October 1983, this virus had become virulent (vir), causing up to 80% mortality in domestic poultry (1, 2). Gene reassortment studies established that the difference between the viruses was a mutation of the hemagglutinin (HA) (3), a major surface glycoprotein responsible for viral attachment to, and penetration of, host cells. Nucleotide sequencing analysis suggested that the critical mutation eliminated a possible site for the attachment of an N-linked oligosaccharide at Asn-11 of HAl of the avirulent virus by altering a sequence for glycosylation. Preliminary evidence based on molecular weight differences in HAl and growth in the presence of tunicamycin suggested loss of a carbohydrate (4). However, since not all Asn-XaaSer/Thr sites are glycosylated in different influenza virus HAs, we do not know how many of the potential glycosylation sites contain carbohydrate, and since all of the influenza virus HAs studied to date contain a carbohydrate at this location in the HA molecule (5, 6), the question of glycosylation at this site remained unresolved.The HA protein of influenza viruses is post-translationally modified by cleavage at a connecting peptide region into subunits HA1 and HA2 (7). This cleavage is a prerequisite for viral infectivity (8), and a correlation has been drawn between HA cleavage and virus production in tissue culture and the virulence of the strain (9-11). Although exogenously added trypsin can cleave the HA and permit infectivity in vitro, the mechanism by which HA is processed and cleaved in vivo is unknown.To determin...

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mentioning

confidence: 99%

Glycosylation affects cleavage of an H5N2 influenza virus hemagglutinin and regulates virulence.

Deshpande¹,

Fried²,

Ando³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

197

132

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Fluorescence in Organized Assemblies

Hinze¹

2000

Encyclopedia of Analytical Chemistry

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This article provides an overview of the general properties of organized assembly (ordered media) systems such as aqueous surfactant and bile salt micelles, lipid and surfactant vesicles (liposomes) and cyclodextrins (CDs) and summarizes their utilization to enhance the performance of analytical fluorescence measurements. In many instances, organic molecules and metal complex species, when included within a CD cavity or solubilized and bound to surfactant aggregates, exhibit enhanced fluorescence. This gives rise to improved detectability of such analytes. The altered microenvironment within the organized medium is capable of impeding the interfering action of other species (inorganic or organic) present in the sample matrix. This often can improve the selectivity of the analytical method. These benefits of improved sensitivity and selectivity arise from the compartmentalization, isolation and shielding of the excited singlet state of the guest analyte from quenching and nonradiative decay processes as well as prevent side reactions that otherwise can occur in bulk solution (or sample matrix). In addition, organic solvents or time‐consuming extraction steps can be avoided owing to the increased solubility of the nonpolar organic or inorganic reagents and/or analyte molecules in water in the presence of the organized medium; allowing for the use of an aqueous medium to perform the procedure. The possibility of conducting reactions and forming fluorescent organic or metal chelates in micellar (or other organized) media that are not observed in a bulk homogeneous solvent system serves to expand the scope of chemistries that one can consider using to design/develop new, unique and improved fluorescent assays. Numerous representative examples of fluorescent methods for determination of both organic and inorganic analytes are provided which serve to illustrate the advantages and benefits accrued from the use of the micelles, vesicles, liposomes or CDs in such procedures. Some experimental considerations and cautions to keep in mind when utilizing organized media are also delineated. An extensive reference section is provided so that the interested reader can easily refer to such for more detailed information on these systems and topics.

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Fluorescence in Organized Assemblies

Memon¹,

Balouch²,

Hinze³

2008

Encyclopedia of Analytical Chemistry

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This article provides an overview of the general properties of organized assembly (ordered media) systems, such as aqueous surfactant and bile salt micelles, lipid (liposomes) and surfactant vesicles, and cyclodextrins (CDs), and summarizes their utilization to enhance the performance of analytical fluorescence measurements. In many instances, organic molecules and metal complex species when included within a CD cavity or solubilized and bound to surfactant aggregates exhibit enhanced fluorescence, improving detectability of such analytes. The altered microenvironment within the organized medium can impede the interfering action of other species (inorganic or organic) present in the sample matrix, which often improves the selectivity of the analytical method. These benefits of improved sensitivity and selectivity arise from the compartmentalization, isolation, and shielding of the excited singlet state of the guest analyte from quenching and nonradiative decay processes, as well as preventing side reactions that otherwise can occur in bulk solution (or sample matrix). Organic solvents or time‐consuming extraction steps can also be avoided, owing to the increased solubility of nonpolar organic or inorganic reagents and/or analyte molecules in water in the presence of the organized medium, allowing the use of an aqueous medium to perform the procedure. The possibility of conducting reactions and forming fluorescent organic or metal chelates in micellar (or other organized) media that are not observed in a bulk homogeneous solvent system serves to expand the scope of possible chemistries for design/development of new, unique, and improved fluorescent assays. Examples of fluorescent methods for determination of both organic and inorganic analytes are provided, which serve to illustrate the advantages and benefits accrued from the use of the micelles, vesicles, liposomes, or CDs in such procedures, along with experimental considerations and cautions in utilizing organized media.

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Protein quantitation at the picomole level: An o-phthaldialdehyde-preTSK column-derivatization assay

Cited by 21 publications

References 5 publications

Glycosylation affects cleavage of an H5N2 influenza virus hemagglutinin and regulates virulence.

Glycosylation affects cleavage of an H5N2 influenza virus hemagglutinin and regulates virulence.

Fluorescence in Organized Assemblies

Fluorescence in Organized Assemblies

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