Purification of insect hyperglycaemic and heart accelerating hormones

Extracts of Periplaneta americana corpora cardiaca were subjected to separation procedures on columns. The eluates were assayed biologically on the cockroach for heart accelerating and hyperglycaemic action. In order to determine the exact location of the peaks, the recovery with respect to the crude extract, and the degree of purification obtained, quantitative assays were done on the basis of arbitrarily established activity units. Gel filtration through columns of Sephadex G-25 was found to separate the hyperglycaemic from the heart-accelerating activity in two distinct peaks, located in the zone expected for peptides. Reasonably good recoveries were obtained with respect to the activity of the crude extract. No activity was found in the zone where a pure sample of 5-hydroxytryptamine was found to emerge. The hyperglycaemic material recovered from this column could be separated in two different peaks by chromatography on a column of SE-Sephadex C-50. An extract of whole cockroach heads was fractionated by gel filtration first on a column of Bio-gel P-10, then on a column of Sephadex G-25. As a result of this procedure the hyperglycaemic activity was purified 61 times, and the heart accelerating activity 98 times with respect to the crude extract (purification was estimated from increase in activity units/mg of protein). Gel filtration through Sephadex G-25 was employed to fractionate an extract of whole heads of Apis mellifica . The higher molecular weight material showed both hyperglycaemic and heart-accelerating activity on cockroach preparations.