Improved method for the isolation and preliminary characterization of human DAF (decay-accelerating factor).

Area of Science:

• Biochemistry
• Immunology
• Cell Biology

Background:

• Decay-accelerating factor (DAF) is an integral erythrocyte membrane protein regulating complement activation.
• Purification of DAF has been challenging due to co-purification with glycophorin.

Purpose of the Study:

• To develop an effective method for removing glycophorin and purifying DAF.
• To characterize the purified DAF.

Main Methods:

• Limited trypsinization of erythrocyte stromata to digest glycophorin.
• DEAE-Sephacel and Bio-Gel A 0.5 m chromatography for DAF extraction.
• SDS-PAGE and amino acid analysis for DAF characterization.

Main Results:

• A novel method effectively removed glycophorin through limited trypsinization.
• Purified DAF exhibited a molecular weight of approximately 70 kDa on SDS-PAGE.
• Amino acid analysis revealed high serine and glutamic acid content; N-terminal sequencing showed no homology with glycophorin.

Conclusions:

• Limited trypsinization is an effective strategy for glycophorin removal.
• The developed method allows for the purification of DAF free from glycophorin contamination.
• Characterization confirms the identity and properties of the purified DAF.