TY - JOUR
T1 - Improvement of heme oxygenase-1-based heme sensor for quantifying free heme in biological samples
AU - Taira, Junichi
AU - Nakashima, Yukinori
AU - Yoshihara, Shun
AU - Koga, Shinya
AU - Sueda, Shinji
AU - Komatsu, Hideyuki
AU - Higashimoto, Yuichiro
AU - Takahashi, Toru
AU - Tanioka, Nohito
AU - Shimizu, Hiroko
AU - Morimatsu, Hiroshi
AU - Sakamoto, Hiroshi
N1 - Funding Information:
This work was supported in part by a Grant-in-Aid for Scientific Research (C) ( 24510301 to H. Sakamoto) from the Japan Society for the Promotion of Science , a grant from Takeda Science Foundation (to J. Taira), and a Grant-in Aid for Young Scientists (B) ( 25870990 to J. Taira) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan .
Publisher Copyright:
© 2015 Elsevier Inc. All rights reserved.
PY - 2015/11/15
Y1 - 2015/11/15
N2 - We recently reported a novel heme sensor using fluorescently labeled heme oxygenase-1; however, its inherent enzyme activity would be a potential obstacle in quantifying heme in biological samples. Here, we found that mutation of the catalytically important residue, Asp140, with histidine in the sensor not only diminished the heme degradation activity but also increased heme binding affinity. The sensor with a visible fluorophore was also found to be beneficial to avoid background emission from endogenous substance in biological samples. By using the improved heme sensor, we succeeded in quantifying free heme in rat hepatic samples for the first time.
AB - We recently reported a novel heme sensor using fluorescently labeled heme oxygenase-1; however, its inherent enzyme activity would be a potential obstacle in quantifying heme in biological samples. Here, we found that mutation of the catalytically important residue, Asp140, with histidine in the sensor not only diminished the heme degradation activity but also increased heme binding affinity. The sensor with a visible fluorophore was also found to be beneficial to avoid background emission from endogenous substance in biological samples. By using the improved heme sensor, we succeeded in quantifying free heme in rat hepatic samples for the first time.
KW - Biological sample
KW - Fluorescent labeling
KW - Free heme
KW - Heme degradation
KW - Heme oxygenase-1
UR - http://www.scopus.com/inward/record.url?scp=84942032064&partnerID=8YFLogxK
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U2 - 10.1016/j.ab.2015.08.004
DO - 10.1016/j.ab.2015.08.004
M3 - Article
C2 - 26278172
AN - SCOPUS:84942032064
SN - 0003-2697
VL - 489
SP - 50
EP - 52
JO - Analytical Biochemistry
JF - Analytical Biochemistry
ER -