Improvement of heme oxygenase-1-based heme sensor for quantifying free heme in biological samples.
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.
Department of Bioscience and Bioinformatics, Graduate School of Computer Science and Systems Engineering, Kyushu Institute of Technology, Kawazu, Iizuka 820-8502, Japan.
Junichi
J
Taira
Department of Bioscience and Bioinformatics, Graduate School of Computer Science and Systems Engineering, Kyushu Institute of Technology, Kawazu, Iizuka 820-8502, Japan.
Yukinori
Y
Nakashima
Department of Bioscience and Bioinformatics, Graduate School of Computer Science and Systems Engineering, Kyushu Institute of Technology, Kawazu, Iizuka 820-8502, Japan.
Shun
S
Yoshihara
Department of Bioscience and Bioinformatics, Graduate School of Computer Science and Systems Engineering, Kyushu Institute of Technology, Kawazu, Iizuka 820-8502, Japan.
Shinya
S
Koga
Department of Bioscience and Bioinformatics, Graduate School of Computer Science and Systems Engineering, Kyushu Institute of Technology, Kawazu, Iizuka 820-8502, Japan.
Shinji
S
Sueda
Department of Bioscience and Bioinformatics, Graduate School of Computer Science and Systems Engineering, Kyushu Institute of Technology, Kawazu, Iizuka 820-8502, Japan.
Hideyuki
H
Komatsu
Department of Chemistry, Kurume University School of Medicine, Asahi-machi, Kurume 830-0011, Japan.
Yuichiro
Y
Higashimoto
Faculty of Health and Welfare Science, Okayama Prefectural University, Kuboki, Okayama 719-1197, Japan; Department of Anesthesiology and Resuscitology, Okayama University Medical School, Shikatacho, Okayama 700-8558, Japan.
Toru
T
Takahashi
Department of Anesthesiology and Resuscitology, Okayama University Medical School, Shikatacho, Okayama 700-8558, Japan.
Nohito
N
Tanioka
Department of Anesthesiology and Resuscitology, Okayama University Medical School, Shikatacho, Okayama 700-8558, Japan.
Hiroko
H
Shimizu
Department of Anesthesiology and Resuscitology, Okayama University Medical School, Shikatacho, Okayama 700-8558, Japan.
Hiroshi
H
Morimatsu
Department of Bioscience and Bioinformatics, Graduate School of Computer Science and Systems Engineering, Kyushu Institute of Technology, Kawazu, Iizuka 820-8502, Japan. Electronic address: sakakan@bio.kyutech.ac.jp.
Hiroshi
H
Sakamoto
D000085:Acetates / Q000737:chemistry; D019943:Amino Acid Substitution; D000818:Animals; D015374:Biosensing Techniques; D020134:Catalytic Domain; D002867:Chromones / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006418:Heme / Q000032:analysis* / Q000378:metabolism; D006419:Heme Oxygenase (Decyclizing) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007564:Japan; D007700:Kinetics; D008862:Microsomes, Liver / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012235:Rhodamines / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013451:Sulfonic Acids / Q000737:chemistry*; D017186:Titrimetry
D000085:Acetates; C400304:Alexa 350; C000608607:Alexa Fluor 555; D002867:Chromones; D005456:Fluorescent Dyes; D050505:Mutant Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012235:Rhodamines; D013451:Sulfonic Acids; D006418:Heme; D006419:Heme Oxygenase (Decyclizing); C491523:Hmox1 protein, rat; D003545:Cysteine
Biological sample; Fluorescent labeling; Free heme; Heme degradation; Heme oxygenase-1