Determination of the internal volume of reconstituted sendai virus envelopes by quenching of calcein fluorescence.
Sendai virus envelopes (SVE) were isolated from Sendai virus particles by Triton X-100 solubilization and ultracentrifugation. The envelopes were reconstituted in the presence of the fluorescent dye calcein by gradual removal of the detergent with Bio-beads SM-2. The internal volume of reconstituted Sendai virus envelopes (RSVE) was determined by quenching the fluorescence of calcein with cobalt (II) ions. The internal volume of RSVE was found to be proportional to the initial SVE protein concentration in the reconstitution mixture, reaching about 18% of the total volume with 5.6 mg of SVE protein per ml. When radiolabelled cloned Epstein-Barr virus DNA fragment was included in the reconstitution mixture, the proportion of DNA associated with the vesicles much exceeded the trapping volume, indicating adsorption of DNA to the internal surface of RSVE. These determinations will allow optimization of the use of RSVE as gene-transfer vehicles.
D004279:DNA, Viral / Q000378:metabolism; D005452:Fluoresceins; D005822:Genetic Vectors; D010222:Parainfluenza Virus 1, Human / Q000235:genetics / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000378:metabolism*
D004279:DNA, Viral; D005452:Fluoresceins; D014759:Viral Envelope Proteins; C007740:fluorexon