JBC, Vol. 252, Issue 7, 2390-2395, Apr, 1977
Preparation and properties of immobilized rubredoxin
W. May and J. Y. Kuo
Rubredoxin, one of the three protein components of the
epoxidation/hydroxylation system of Pseudomonas oleovorans was immobilized
by attachment to CNBr-activated agarose (Sepharose 4B). Since this
represents the first reported example of the preparation of a
water-insoluble derivative of an enzyme of this type, the electron transfer
and physical properties of the conjugate were examined in order to allow
comparison with those of the soluble enzyme. Immobilized rubredoxin
exhibits all of the major spectral properties of the soluble enzyme above
300 nm, but some distortion in the 280 nm abosrbance band was observed. The
immobilized enzyme accepts electrons from dithionite or form NADPH in the
presence of spinach ferredoxin-NADP reductase, and upon reduction the
visible absorbance is bleached. Immobilized rubredoxin mediates the
reduction of cytochrome c in the presence of NADPH and spinach reductase,
although it is less efficient in this role than soluble rubredoxin. The
oxidation-reduction potential of immobilized rubredoxin was determined and
found to be similar to that of the soluble enzyme. In the presence of 2.5 m
guanidine HCL, the immobilized enzyme is considerably more stable than
soluble rubredoxin toward denaturation. After anaerobic reduction, iron was
readily removed from immobilized rubredoxin by washing in 0.5 m Tris base,
PH 9.5 containing 0.07 M mercaptoethanol, and the resulting immobilized
apoenzyme could then be reconstituted to give back a conjugate with the
original iron content, as judged from its absorbance at 497 NM. Reptition
of the entire reduction-dissociation-reconstitution cycle gave the same
results as were obtained after the initial reconstitution.
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