Conversion of δ-Amino Laevulinic Acid to Porphobilinogen by Different Tissues of the Polychaete Annelid Neoamphitrite figulus

Abstract

THE heart-body, a small spongy mass of tissue within the dorsal vessel of the terebellid polychaete Neoamphitrite figulus (Dalyell), is almost certainly concerned with synthesis of the haemoglobin of the blood. The coelomocytes also contain haemoglobin, but this is different. Kennedy and Dales1 have shown that the heart-body tissue and the coelomocytes in this species converted porphobilinogen (PBG) to uroporphyrin. Tissues were incubated with purified PBG suspended in sea water and at room temperature for varying periods and the products identified after extraction and purification by paper chromatography. Some similarly performed experiments using δ-amino laevulinic acid (ALA) did not give clear results. Subsequent work, using the method of McRae2, whereby the conversion of ALA to PBG is identified against suitable controls by the positive reaction PBG gives with Ehrlich's p-aminobenzaldehyde reagent3, has, nevertheless, given quite clear results. McRae's method was followed in detail except that to quantify the results the tissues were merely dried with acetone and weighed, for it was desired only to compare the relative ability of different tissues to convert ALA to PBG. McRae used homogenates of whole animals (Dugesia). Tissues were deep frozen and homogenized on thawing in M/15 phosphate buffer, pH 7.0, the ALA being added to provide a concentration of 400 µg/ml. of homogenate. Homogenates, suitably controlled, were incubated at 37° C for 2.5 h. The colour of the Ehrlich reaction was measured at 553 mµ after 15 min with a ‘Unicam SP 500’ spectrophotometer. While the activity of each tissue is expressed in arbitrary units, their relative activities in one typical experiment are shown in Table 1. The experiment was repeated five times with similar results. The tissues of five worms were pooled in each experiment. Table 1 shows that the heart-body is ten times more active than any other tissue determined. The body wall muscle is colourless and if it contains any myoglobin this is in a relatively low concentration and any ALAase present is insufficient to produce enough PBG to be detected by the method. On the other hand, Kennedy and Dales4 detected coproporphyrin III, which must have been formed during haem synthesis, in the body wall; whether this is formed in the skin or is deposited there, as this negative result suggests, is being investigated further. The hind stomach or gizzard has muscles of a deep red colour and some ability to convert ALA to PBG is not surprising. The ability of the coelomocytes to do so is also expected, confirming the hypothesis that they synthesize their own haemoglobin from ALA and do not receive compounds later in the biosynthetic pathway to haem. The high activity of the gut (fore stomach plus intestine) from which the contents had carefully been removed is surprising, especially because Kennedy and Dales4 found no free porphyrins in these tissues. It may be that the coelomocytes, which contain haemoglobin, arise from the coelomic epithelium of the gut wall. This is being investigated further.