Electrophoretic phenotyping of erythrocyte enzymes

Abstract Erythrocyte acid phosphatase (EAP), esterase D (ESD) and phosphoglucomutase (PGM) phenotypes among the erythrocyte enzyme types of blood groups are surveyed and a modified cellulose acetate membrane isoelectric focusing (CAM-IEF) method for their exploration is described. The phenotyping procedures are usually classified as either equilibrium or non-equilibrium IEF. Equilibrium IEF, which is based on differences in p I values, includes three methods: (i) a narrow pH range of carrier ampholytes, (ii) a relatively narrow pH range of carrier ampholytes containing chemical separators and (iii) immobilized pH gradient gels. Among the three methods, immobilized pH gradients provide a better resolution of isozymes. Conversely, the disadvantages of immobilized pH gradients include longer focusing times and complex gel preparations. Moreover, immobilized pH gradients are unsuitable for stain analysis because of the insensitivity of PGM1 detection. A hybrid IEF system and a commercial immobilized pH gradient dry plate have overcome these problems. However, EAP typing is extremely expensive and ESD typing is not well distinguished by hybrid IEF. As each method has both merits and demerits, the most suitable technique should be selected based on the kind of erythrocyte enzyme types and sample conditions. On the other hand, non-equilibrium IEF is a rapid method because isozymes are detected on the basis of their charge differences under non-equilibrium conditions. Moreover, the appropriate addition of chemical separators increases the charge difference and provides a good resolution within a shorter time. Addition of more separators produces a narrow pH range in the gel and takes a substantially longer time to reach the optimum pH range for charge difference. Further, the viscosity due to the excess addition of separators results in poor reproducibility for high-field-strength CAM-IEF. Hence the amount of chemical separators is taken into consideration of obtaining optimum results. If the optimum conditions are established with primary experiments, the combination of chemical separator and non-equilibrium IEF is proposed as a readily available method for routine analysis of erythrocyte enzyme types in forensic science.