Improved esterase D typing on ultrathin isoelectric focusing gels using narrow interval Pharmalyte carrier ampholytes (pH 4.5–5.4) in conjunction with a separator and a thickness modified pH gradient

Abstract

Home Office Forensic Science Service Aldermaston. Reading, Berkshire -, RG7 4PNand a thickness modified pH gradient Three common alleles are found in esterase D: EsD 1, EsD 2 and EsD 5. In addition, a number of rare variants are found. The isoelectric points of the EsD 1 and EsD 2 isoenzymes differ by only 0.02 pH units, which could lead to possible confusion between the two using conventional isoelectric focusing (IEF). Therefore, in order to ensure adequate separation of these two bands, it is necessary to use a narrow pH gradient. The separation of esterase D (EsD) has been improved on electrofocusing gels by incorporating a narrow range Pharmalyte (pH 4.5-5.4) in conjunction with the separator N~(2-hydroxyethyl)piperazine-N'-2-ethanesulphonic acid (HEPES). This mixture resultedintheformationofanarrowpHrangeofpH 4.85-5.35 (themajor typing bands of EsD fall within the range of p l 4.9-5.2). Resolution was further improved by the use of thickness modified pH gradients in ultrathin gels which flattened the pH gradient in the range ofpH 5.0-5.1. The techniques used were shown to be effective for typing bloodstains up to 1 month old.

Forensic science

“…The simultaneous isoelectric focusing of EAP and EsD was accomplished and applied to bloodstains (273). EsD has been typed by isoelectric focusing (274)(275)(276) as was erythrocyte acid phosphatase (EAP) (277). A method using a double origin sample application system was developed for the simultaneous separation of ADA, AK, and carbonic anhydrase II by agarose gel electrophoresis (278).…”