C O M M U N I C A T I O N S
and subsequently washing the cells to remove extracellular Cbl prior
•-
to paraquat exposure resulted in intracellular O
2
levels returning to
normal. The same effect was observed with SOD itself (Figure 3).
Further details are given in the SI.
To summarize, we have shown that cob(II)alamin, an important
•
-
intracellular Cbl form, reacts rapidly with O
2
at rates approaching
8
9
-1 -1
those of SOD (7 × 10 versus 2 × 10 M s , respectively). This
•-
suggests that direct scavenging of O
2
is an important molecular
mechanism by which Cbl modulates intracellular signal transduction
and protects against chronic inflammation. Cbl likely acts as a second
•-
Figure 2. Competition kinetics with Cu,Zn-SOD. (A) Plot of change of
line of defense when O
levels overwhelm the SOD protection
2
absorbance at 351 nm (dashed line) versus time for the oxidation of Cbl(II)
system, perhaps accounting for the significantly increased oxidative
damage markers in patients with inherited disorders of intracellular
•
-
•-
(
5
50 µM) by O
2
(O
2
flux ) 10 µM/min; 1000 U/ml catalase, 1 mM EDTA,
0 mM NaCl, 10 mM phosphate buffer, pH 7.4, 25.0 °C). The experiment
19
was repeated with increasing SOD (2.0-88 µM, solid lines; arrow indicates
Cbl metabolism. Given that B12 is nontoxic even at high doses and
that a significant proportion of the elderly are B12 deficient, our results
provide a compelling argument for clinical trials studying the
pharmacological effects of B12 in the treatment and prevention of
diseases associated with chronic inflammation and aging.
increasing SOD concentration). The rate of Cbl(II) autoxidation is also shown
(
)
dotted line). (B) Plot of V
(6.8 ( 0.8) × 10 M
0
/VSOD - 1 vs [SOD]. The best fit to eq 1 gave kCbl
8
-1 -1
s .
that increased scatter in the data occurs at high SOD concentrations
as expected, given that the plots use the ratio V /VSOD, which has a
0
Acknowledgment. The authors thank Dr. B. Alvarez and Dr. G.
Ferrer-Sueta for useful discussions and Dr. D. Jacobsen and Dr. I.
Batini c´ -Haberle for the HAEC and MnTEPyp, respectively. This work
was supported by the American Heart Association (Predoctoral grant,
E.S.M), the NSF (CHE-0848397, N.E.B.), the Ohio Board of Regents
larger relative error at smaller VSOD values (i.e., high [SOD]). The best
8
-1 -1
fit of the data at 351 nm gave kCbl ) (6.8 ( 0.8) × 10 M
s (Figure
8
-1
2
B). Similar experiments at 474 nm gave kCbl ) (8 ( 1) × 10 M
-1
s
(Figure S4). The smaller ∆Αbs at 474 nm results in a less reliable
value of kCbl
.
(J.Y.), and KSU GSS (E.S.M.).
Importantly, since XO requires oxygen for its activity, the experi-
ments were conducted in an aerobic buffer. However, Cbl(II) undergoes
slow disproportionation to cob(I)alamin (Cbl(I)) and Cbl(III) ()
Supporting Information Available: Experimental section; derivation
of eq 1; Figures S1-S5, Table S1 and S2. This material is available free
of charge via the Internet at http://pubs.acs.org.
+
H
2
OCbl /HOCbl) in air, followed by rapid oxidation of Cbl(I) to
15
Cbl(III). The rate of Cbl(II) autoxidation in the absence of XO was
therefore determined and subtracted from the V and VSOD data (dotted
line in Figure 2A). The reaction between H OCbl /HOCbl and O
to form superoxocobalamin (this species has been characterized at low
0
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#
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JA904670X
J. AM. CHEM. SOC. 9 VOL. 131, NO. 42, 2009 15079