45(20 μg/ml) is as threefold as that in bacteria treated by RIF (20 μg/ml) + ART
(60 μg/ml).
It means that enhanced H 2 O 2 production following CAT inhibition by ART
should facilitate the bactericidal effect of antibiotics. Therefore, ART can abrogate
the protective NO, on the one hand, and also promote the harmful H 2 O 2 , on the
other hand, thereby exhibiting a dual synergistic effect as the inhibitors of both
bNOS and CAT.
4.2.2.4 Identification of ART-Bacterial Heme Conjugates
Based on our assumption, ART would bind to bNOS, CAT, and other hemopro-
teins, hence prohibiting the interconversion between Fe^3 + and Fe^2 +. We monitored
the dynamic fluctuations of heme and ART-heme conjugates. As results, A 415 that
reflects the absorbance of heme and A 476 that represents the absorbance of ART-
heme conjugates were observed to have higher readings (A 415 = 0.3; A 476 = 0.2)
after adding ART for three hours. As comparison, the lower readings (A 415 = 0.18;
A 476 = 0.06) were determined in bacterial cultures without ART supplementation.
After elevations by three-hour incubation, both A 415 and A 476 decline to readings
equal to the control by incubation for 6–9 h.
The elevation of A 415 was assumed to the de novo biogenesis of hemoproteins,
while the elevation of A 476 could be a hint indicating the formation of ART-heme
conjugates. The increases of hemoproteins including bNOS and CAT need the over-
expression of corresponding genes, because they should be inducible after the inacti-
vation of those enzymes due to heme alkylation by ART. The double decline of A 415
and A 476 is likely attributed to bacterial death upon ART’s bactericidal effects.
Fig. 4.2 CAT activity in B. Licheniformis upon exposure to RIF or ART + RIF. ART arte-
misinin; Rif rifampicin. A single asterisk (*) represents significant difference in ART + RIF from
RIF (P < 0.05); Double asterisks (**) represent very significant difference in ART + RIF from
RIF (P < 0.01)
4.2 In Vitro Examination for ART Suppressing ...