Absorbance spectra and Hb degradation

We use absorbance spectra to follow the effects of superoxide and hydrogen peroxide on the Hb components of PolyHb and PolyHb-CAT-SOD (D'Agnillo & Chang, 1998a) (Fig. 4.6). Xanthine-xanthine oxidase is used to generate superoxide (Fig. 4.12 ABC). For PolyHb, this results in a stepwise decrease in absorbance at 540 and 577 nm, showing that Hb (Fe2+) is oxidized to metHb (Fe3+) (Fig. 4.12A). Increase in metHb (Fe3+) is shown as a stepwise increase at the absorbance peak of 630 nm (Fig. 4.6A). PolyHb-CAT-SOD with a SOD/CAT ratio of 0.01 prevents this oxidation (B), while a SOD/CAT ratio of 100 produces no protective effect (Fig. 4.12C). A glucose-glucose oxidase system is used to generate hydrogen peroxide (Fig. 4.12 DEF). The ferryl (Fe4+) intermediate is shown by the appearance of new peaks at 545 and 580nm (Fig. 4.12D). PolyHb-CAT-SOD with a SOD/CAT ratio of 0.01 reduces this oxidation (Fig. 4.5E), while a SOD/CAT ratio of 100 produces no protective effect (Fig. 4.12F).

Fig. 4.12. Absorbance spectra of reaction mixtures containing 50/M of Hb and 100 /M of xanthine, and 10mU/ml of xanthine oxidase (left) or 10 mM of glucose and 10/g/ml glucose oxidase (right). SOD/CAT in each mixture are shown. Each panel represents a series of spectra collected over a period of 20 min at 1 scan/min. In the absence of CAT or even the addition of 3 U/ml of CAT, there is still inadequate protection from the effects of hydrogen peroxide. 300 U/ml of CAT to have significant protective effects. (D'Agnillo & Chang, 1998a.)

Fig. 4.12. Absorbance spectra of reaction mixtures containing 50/M of Hb and 100 /M of xanthine, and 10mU/ml of xanthine oxidase (left) or 10 mM of glucose and 10/g/ml glucose oxidase (right). SOD/CAT in each mixture are shown. Each panel represents a series of spectra collected over a period of 20 min at 1 scan/min. In the absence of CAT or even the addition of 3 U/ml of CAT, there is still inadequate protection from the effects of hydrogen peroxide. 300 U/ml of CAT to have significant protective effects. (D'Agnillo & Chang, 1998a.)

In a separate study (Fig. 4.13), hydrogen peroxide is added to PolyHb (10 mM) or PolyHb-SOD-CAT (10 mM), and the absorbance spectra (450-700 nm) is followed (Fig. 4.13). For PolyHb, the spectral changes reflect the oxidation of ferrous (Fe2+)-heme to ferric (Fe3+)-heme (Fig. 4.13AB). The rate of degradation increases with increase in hydrogen peroxide added in amounts from 10 to 100 to 500ßM. The absorbance spectra of PolyHb-CAT-SOD are minimally affected, indicating that these reactions are minimized due to the elimination of H2O2 (Fig. 4.13CD). Similar results are observed following

PolyHb

Time = 1 min after H2O2 addition

PolyHb

Time = 60 min after H2O2 addition

0.25

PolyHb

Time = 1 min after H2O2 addition

PolyHb

Time = 60 min after H2O2 addition

0.25

PolyHb-SOD-CAT

Time = 1 min after H2O2 addition

PolyHb-SOD-CAT

Time = 60 min after H2O2 addition

PolyHb-SOD-CAT

Time = 1 min after H2O2 addition

Fig. 4.13. Absorbance spectra of PolyHb and PolyHb-SOD-CAT (10/M) following H2O2 addition of 0, 10, 100, and 500/M. (From D'Agnillo & Chang, 1993.)

PolyHb-SOD-CAT

Time = 60 min after H2O2 addition

Wavelength(nm)

Fig. 4.13. Absorbance spectra of PolyHb and PolyHb-SOD-CAT (10/M) following H2O2 addition of 0, 10, 100, and 500/M. (From D'Agnillo & Chang, 1993.)

oxidative challenge with exogenous superoxide (O2-) produced from xanthine/xanthine oxidase.

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