Isolation of influenza virus A hemagglutinin C-terminal domain by hemagglutinin proteolysis in octylglucoside micelles.
A method of isolation of hydrophobic membrane-bound C-terminal domain of influenza virus A hemagglutinin (HA) is suggested. The method is based on the insertion of HA into octylglucoside micelles followed by pepsin or thermolysin hydrolysis. Subsequent treatment of proteolytic digests with chloroform-hexafluoroisopropanol mixture resulted in the extraction of a few hydrophobic peptides into organic phase. Mass-spectrometry (MALDI-TOF) analysis revealed that the peptides with ion masses corresponding to the anchoring C-terminal domain with or without modifications predominated in the organic solution. The data obtained confirmed our speculation on the possibility of the suggested isolation scheme following from the strong interactions of anchoring domains in compact trimeric structure of HA spikes combined with micelle protection effect. Several appropriate peptides presence in the organic phase apparently arises from the presence of a few accessible proteolytic sites in HA transmembrane region.
Radyukhin
Victor A
VA
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Leninskie Gory, Moscow 119992, Russia. varvic@belozersky.msu.ru
Serebryakova
Marina V
MV
Ksenofontov
Alexander L
AL
Lukashina
Elena V
EV
Baratova
Lyudmila A
LA
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D003902:Detergents / Q000737:chemistry*; D005960:Glucosides / Q000737:chemistry*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D009980:Influenza A virus / Q000737:chemistry*; D008823:Micelles; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013820:Thermolysin / Q000378:metabolism
D003902:Detergents; D005960:Glucosides; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D008823:Micelles; D010455:Peptides; C018619:octyl-beta-D-glucoside; D010434:Pepsin A; D013820:Thermolysin