Biochemical Characteristics of Phenotypes

Differences in the electrophoretic patterns and activities between the phenotypes raised the possibility that they might also be characterized in a more specific and detailed biochemical manner. In 1949, Abul-Fadl and King (A4) observed that 0.5% formaldehyde inhibited erythrocyte acid phosphatase completely, whereas 0.01 Af l-( + )-tartrate had no effect. When 0.5% formaldehyde was added to the reaction mixture in gel electrophoresis (H13), it completely inhibited all variants of erythrocyte acid phosphatase, so that no zones of activity in any of the five types were visible after the 3-hour incubation. 

Luffman and Harris (L13) applied several other criteria in an attempt to differentiate among the various phenotypes. Incubation of hemolysates representing these phenotypes showed that at temperatures of 47°C to 52 °C types CA and CB were denatured more slowly than the other types tested. A, BA, and B. For example, after 30 minutes at 50°C, the average losses in activity were 52% for CA and 67% for CB, as compared with losses of 89% for A, 83% for BA, and 80% for B. 

Incubation with guanidine or urea at 28 C for 20 minutes showed denaturation to be dependent on the concentration, but there were no 

The column chromatography of the five common phenotypes were also studied by Hopkinson and Harris (H12). A 10-ml aliquot of the supernatant from a centrifuged hemolysate was applied to the DEAE column which had been washed with Tris-phosphate buffer (pH 8.0). The column was then washed with the starting buffer to elute the hemoglobin. The enzyme was eluted with an exponential gradient of sodium chloride in Tris-phosphate buffer and collected in 2-ml fractions at a flow rate of 20 ml/hour. 

Two distinct peaks of acid phosphatase activity were detected in each phenotype, but the positions of these peaks differed. For example, in phenotype A, the peaks were approximately at tubes 150 and 190 in B, at about 130, 170, and 265 in BA, at 110 and 155. In these three, the first peaks showed minor enzyme activity. In CA, there was a major peak at about tube 130 and a smaller one at about tube 170. The shape of the curves varied according to the phenotype tested. In general, these results confirmed what gel electrophoresis originally showed, namely, that there are charge differences between the various isoenzymes. The electrophoretic patterns may also be influenced by the type of buffer used to make up the starch gel (K2). 

---