Identification of a Spectrally Stable Proteolytic Fragment of Human Neutrophil Flavocytochrome b Composed of the NH2-terminal Regions of gp91phox and p22phox

Abstract

A heme-bearing polypeptide core of human neutrophil flavocytochrome b 558 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 NH 2-terminal 169-171 amino acid residues of p22phox. These findings provide direct evidence that the primarily hydrophobic NH 2-terminal regions of flavocytochrome b are responsible for heme ligation.