posted on 2017-11-21, 00:00authored byQing Chen, Xue Hu, Dan-Dan Zhang, Xu-Wei Chen, Jian-Hua Wang
The
bioconjugation of a polyoxometalate (POMs), i.e., dodecavanadate
(V12O32), to DNA strands produces a functional
labeled DNA primer, V12O32-DNA. The grafting
of DNA primer onto streptavidin-coated magnetic nanoparticles (SVM)
produces a novel composite, V12O32-DNA@SVM.
The high binding-affinity of V12O32 with the
ATP binding site in myosin subfragment-1 (S1) facilitates favorable
adsorption of myosin, with an efficiency of 99.4% when processing
0.1 mL myosin solution (100 μg mL–1) using
0.1 mg composite. Myosin adsorption fits the Langmuir model, corresponding
to a theoretical adsorption capacity of 613.5 mg g–1. The retained myosin is readily recovered by 1% SDS (m/m), giving
rise to a recovery of 58.7%. No conformational change is observed
for myosin after eliminating SDS by ultrafiltration. For practical
use, high-purity myosin S1 is obtained by separation of myosin from
the rough protein extract from porcine left ventricle, followed by
digestion with α-chymotryptic and further isolation of S1 subfragment.
The purified myosin S1 is identified with matrix-assisted laser desorption/ionization
time-of-flight/mass spectrometry, giving rise to a sequence coverage
of 38%.