Purification and partial characterization of the b-type cytochrome from human polymorphonuclear leukocytes

Area of Science:

• Biochemistry
• Cell Biology
• Immunology

Background:

• Polymorphonuclear leukocytes possess an oxidase system crucial for generating reactive oxygen species like superoxide radicals and hydrogen peroxide.
• A non-mitochondrial cytochrome b is implicated in this oxygen species generation pathway within phagocytes.

Purpose of the Study:

• To purify and characterize a non-mitochondrial cytochrome b from human polymorphonuclear phagocytes.
• To investigate the biochemical and spectral properties of the purified cytochrome b.

Main Methods:

• Solubilization of cytochrome b using Triton X-100.
• Chromatography on Blue Sepharose and Sephacryl S-300 for purification.
• Gel filtration and SDS-PAGE for molecular mass determination.
• Spectrophotometry for spectral analysis and midpoint potential determination.

Main Results:

• The cytochrome b was purified 170-fold with a yield of 21%.
• The purified protein consists of a single 127 kDa polypeptide with an estimated native molecular mass of 235 kDa.
• Spectral analysis revealed characteristic absorbance bands, and carbon monoxide binding was time-dependent.
• Purified cytochrome b exhibited a significantly decreased midpoint potential (-407 mV) compared to its in situ state (-218 mV).

Conclusions:

• A homogeneous preparation of non-mitochondrial cytochrome b was obtained from human polymorphonuclear phagocytes.
• The purified cytochrome b possesses distinct spectral and electrochemical properties, including a lower midpoint potential than its native form.
• These findings contribute to understanding the role of cytochrome b in the phagocyte oxidase system.