Electrokinetic Analysis of the Fetal Erythrocyte Membrane After Trypsin Digestion

Abstract

Summary: Treatment of intact erythrocytes with trypsin (TRY) produced a significant difference (P < 0.001) in electrophoretic mobility (EM) of fetal and adult cells. The greater net negative charge of the fetal red blood cells (RBC) at the “new electrokinetic surface” generated by TRY was further analyzed with cell-electrophoretic measurements combined with various enzymatic and chemical reactions.

Formaldehyde or diazonium reaction known to eliminate positive (NH₂) groups increased the net negativity of the TRY-digested adult and fetal RBC in the same extent leaving the distance between the EM values unchanged. When the TRY digestion was followed by neuraminidase (NASE) treatment, the decrease in EM of both types of erythrocytes was of the same magnitude, and the distance between the EM values of the TRY-treated fetal and adult RBC remained unchanged, too. Phospholipase-C (PLC) digestion performed after TRY treatment did not cause any change in EM, either in adult or in fetal RBC.

The results show that the greater net negativity of the fetal RBC surface after TRY digestion can be attributed to neither less positive (NH₂) groups, nor more sialic acid or phosphoric acid molecules existing at the new electrokinetic surface. It is suggested that the fetal RBC membrane may possess more acidic amino acid exposed to the outside, when TRY liberates sialoglycoprotein from the outer surface of the membrane.

Speculation: Glycophorin seems to be the only major membrane protein in RBC which is readily accessible for TRY at the exterior cell surface. The new electrokinetic surface generated by TRY is characterized by the portion of glycophorin just behind the TRY splitting site. The results presented here might be explained by a supposed modification in the biogenesis of the glycophorin in fetal life. Amino acid analysis of the glycophorin fragments obtained with TRY digestion would serve a more intimate insight to the problem and would give information about the maturation from fetal to adult surface properties.

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