Analytical titration curves of glycosyl hydrolase Cel45 by combined isoelectric focusing-electrophoresis.
The electrical charge of endocellulase Cel45-core has been determined by combined isoelectric focusing-electrophoresis in the range of pH 3-9. In order to transform electrophoretic mobility to absolute electrical charge value, several corrections were applied: the frictional coefficient theoretically calculated from the molecular dimensions depends on porous gel structure and on the ionic strength of the solution. By comparing the curve calculated according to the Linderstrom-Lang equation, the number of charged electrical groups exposed to the solvent and their apparent ionization constants, pK(o)i, can be determined. Furthermore, the macromolecule structure can be assumed not to change in this pH range. This finding is necessary to understand the structure and the electrical properties of the entire Cel45 molecule.
Attanasio
F
F
Istituto di Studi Chimico Fisici di Macromolecole Sintetiche e Naturali, CNR, Genova, Italy.
Bruschi
M
M
Candiano
G
G
Galletto
R
R
Musante
L
L
Schülein
M
M
Rialdi
G
G
D001230:Aspergillus / Q000201:enzymology; D020134:Catalytic Domain; D002480:Cellulase / Q000032:analysis*; D002482:Cellulose / Q000378:metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005656:Fungal Proteins / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000032:analysis*; D007525:Isoelectric Focusing / Q000295:instrumentation* / Q000379:methods*; D008962:Models, Theoretical; D013696:Temperature; D017186:Titrimetry / Q000295:instrumentation* / Q000379:methods*
D005656:Fungal Proteins; D002482:Cellulose; D006867:Hydrolases; C097231:endoglucanase V; D002480:Cellulase