Physical properties and subunits of Haemopis grandis erythrocruorin

Abstract

The erythrocruorin of the leech Haemopis grandis possessed an s 20,w of 57 S at neutral pH, its isoelectric point at pH 6.0 and exhibited a slightly sigmoid oxygenation curve with n ≈ 2.1 and P 50 = 11.2 mm at pH 7.4. A minimum molecular weight of 24 000 ± 1 500 per heme group was determined from the iron and heme contents, 0.22 ± 0.01 and 2.73 ± 0.14 weight %, respectively. The subunit composition of the erythrocruorin was investigated using gel filtration in sodium dodecyl sulfate and polyacrylamide gel electrophoresis in sodium dodecyl sulfate at neutral pH. Haemopis erythrocruorin dissociated in the presence of sodium dodecyl sulfate into four subunits (1 through 4) possessing molecular weights of about 27 000, 23 000, 21 000 and 13 500, respectively. When the erythrocruorin was reduced with mercaptoethanol prior to sodium dodecyl sulfate electrophoresis, three subunits were observed, possessing molecular weights of about 13 000 (I), 16 500 (II) and 28 000 (III). Sodium dodecyl sulfate electrophoresis of the isolated subunits 1 through 4 showed that subunit I was provided by subunits 1 and 4, subunit II was provided by subunit 1 and subunit III was provided by both subunit 2 and subunit 3. Haemopis erythrocruorin thus appeared to consist of at least five different polypeptide chains. It is likely that not all of the constituent polypeptide chains were associated each with a heme group. The shape of the Haemopis erythrocruorin observed by electron microscopy appeared to be consistent with the two-tiered hexagonal array characteristic of annelid erythrocruorins and chlorocruorins.