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Originally published In Press as doi:10.1074/jbc.M104373200 on August 14, 2001

J. Biol. Chem., Vol. 276, Issue 42, 38852-38861, October 19, 2001
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Identification of a Spectrally Stable Proteolytic Fragment of Human Neutrophil Flavocytochrome b Composed of the NH2-terminal Regions of gp91phox and p22phox*

Thomas R. Foubert, Justin B. Bleazard, James B. Burritt, Jeannie M. Gripentrog, Danas Baniulis, Ross M. Taylor, and Algirdas J. JesaitisDagger

From the Department of Microbiology, Montana State University, Bozeman, Montana 59717-3520

A heme-bearing polypeptide core of human neutrophil flavocytochrome b558 was isolated by applying high performance, size exclusion, liquid chromatography to partially purified Triton X-100-solubilized flavocytochrome b that had been exposed to endoproteinase Glu-C for 1 h. The fragment was composed of two polypeptides of 60-66 and 17 kDa by SDS-polyacrylamide gel electrophoresis and retained a native heme absorbance spectrum that was stable for several days when stored at 4 °C in detergent-containing buffer. These properties suggested that the majority of the flavocytochrome b heme environment remained intact. Continued digestion up to 4.5 h yielded several heme-associated fragments that were variable in composition between experiments. Digestion beyond 4.5 h resulted in a gradual loss of recoverable heme. N-Linked deglycosylation and reduction and alkylation of the 1-h digestion fragment did not affect the electrophoretic mobility of the 17-kDa fragment but reduced the 60-66-kDa fragment to 39 kDa. Sequence and immunoblot analyses identified the fragments as the NH2-terminal 320-363 amino acid residues of gp91phox and the NH2-terminal 169-171 amino acid residues of p22phox. These findings provide direct evidence that the primarily hydrophobic NH2-terminal regions of flavocytochrome b are responsible for heme ligation.

* This work was supported by United States Public Health Service Grant R01 AI 26711 (to A. J. J.) and American Heart Association SDG Award 30156N (to J. B. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Microbiology, Montana State University, 109 Lewis Hall, Bozeman, MT 59717-3520. Tel.: 406-994-4811; Fax: 406-994-4926; E-mail: umbaj@montana.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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