Purification and structural analysis of the fourth component of human complement.

Area of Science:

• Biochemistry
• Immunology
• Proteomics

Background:

• The fourth component of human complement (C4) is crucial for immune responses.
• Understanding C4 structure and function is vital for immunological research.

Purpose of the Study:

• To purify human complement component 4 (C4) from plasma.
• To characterize the subunit structure and inactivation mechanisms of C4.

Main Methods:

• Plasma fractionation using poly(ethylene glycol) precipitation and affinity chromatography to remove plasminogen.
• Sequential ion-exchange chromatography (DEAE-cellulose, QAE-Sephadex, DEAE-Bio-Gel A) for C4 purification.
• Polyacrylamide gel electrophoresis (PAGE) and immunological criteria for homogeneity assessment.
• Dithiothreitol reduction to analyze interchain disulfide bonds and subunit structure.
• Gel filtration in acetic acid for chain separation.
• Automated Edman degradation for N-terminal sequencing.
• Amino acid composition analysis.

Main Results:

• C4 was purified to homogeneity with a 20% yield.
• A three-chain structure (alpha, beta, gamma) was confirmed with estimated molecular weights of 93,000, 75,000, and 30,000 Da, respectively.
• Inactivation by C1s and trypsin fragmented the alpha chain, while hydrazine did not alter chain size.
• N-terminal sequences and amino acid compositions of C4 chains were determined.

Conclusions:

• The study successfully purified and characterized human C4, elucidating its subunit composition.
• Differential fragmentation patterns upon inactivation provide insights into C4's functional mechanisms.
• This work contributes to a deeper understanding of the complement system's role in immunity.