Influence of the methacrylate monolith structure on genomic DNA mechanical degradation, enzymes activity and clogging.
The chromatography of mechanically sensitive macromolecules still represents a challenge. While larger pores can reduce the mechanically induced cleavage of large macromolecules and column clogging, the column performance inevitably decreases. To investigate the effect of pore size on the mechanical degradation of DNA, column permeability and enzyme biological activity, methacrylate monoliths with different pore sizes were tested. Monolith with a 143 nm pore radius mechanically damaged the DNA and was clogged at flow rates above 0.5 ml min(-1) (26 cm h(-1)). For monoliths with a pore radius of 634 nm and 2900 nm, no mechanical degradation of DNA was observed up to 5 ml min(-1) (265 cm h(-1)) above which the HPLC itself became the main source of damage. A decrease of a permeability appeared at flow rate 1.8 ml min(-1) (95 cm h(-1)) and 2.3 ml min(-1) (122 cm h(-1)), respectively. The effect of the pore size on enzyme biological activity was tested with immobilized DNase and trypsin on all three monoliths. Although the highest amount of enzyme was immobilized on the monolith with the smallest pores, monolith with the pore radius 634 nm exhibited the highest DNase biological activity probably due to restricted access for DNA molecules into the small pores. Interestingly, specific biological activity was increasing with a pore size decrease. This was attributed to higher number of contacts between a substrate and immobilized ligand.
Bencina
Katja
K
BIA separations d.o.o., Teslova 30, SI-1001 Ljubljana, Slovenia.
Bencina
Mojca
M
Podgornik
Ales
A
Strancar
Ales
A
D000818:Animals; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D003851:Deoxyribonucleases / Q000378:metabolism*; D016678:Genome / Q000235:genetics*; D008689:Methacrylates / Q000737:chemistry*; D008855:Microscopy, Electron, Scanning; D010539:Permeability; D016062:Porosity; D011312:Pressure
D008689:Methacrylates; D004247:DNA; C427203:calf thymus DNA; D003851:Deoxyribonucleases