Limited Trypsinolysis of β‐Haemocyanin of Helix pomatia

Abstract

A limited trypsinolysis of the tenths of P-haemocyanin of ~e l~x p 5 m a t i u was performed at pH 8.2. The absorbance at 346 nm remained constant, indicating a preservation of the oxygen-binding sites. The five tryptic fragments were separated by chromatography on Sephadex G-100 and on DEAEcellulose. They contained 2 Cu per 50000 daltons and showed different mobilities in agar electrophoresis. The molecular weights indicated that one fragment was constituted of three functional domains of about 50000 daltons, that two fragments were constituted of two domains, and two others of one domain. Twentieths of 8-haemocyanin seemed thus to be made up of 9 domains. The circular dichroic spectra of the fragments indicated the presence of two classes of copper groups according to their positive maximum at 455 or at 500 nm. The circular dichroic spectra also showed that no fragment could have originated from a larger one, confirming the presence of nine domains in the twentieths.

Characterization of domains obtained from a mollusc haemocyanin by limited proteolytic digestion

“…We have not isolated the collar fraction after digestion of one-tenth L. stagiialis molecules, but we identify it as a component of the intermediate peak in gel filtration. It is also possible to conclude that fraction TIC of Gielens et al (1975) is similar to our fraction P3 as judged by the c.d. spectra.…”

“…It has been shown (Hall et al, 1975) that L. stagnalis haemocyanin behaves in some ways as a fl-type haemocyanin. However, the digestion procedure and the fractionation systems used by Gielens et al (1975) were somewhat different from those used in the present work. Indeed, they may be criticised inasmuch as they used Sephadex G-100 for the initial fractionation of digestion products, and also that molecular weights were calculated from sedimentation coefficients by the use of an empirical formula, rather than by sedimentation-equilibrium methods.…”

“…We may compare the findings with L. stagnalis with those of Gielens et al (1975) with H. pomatia fl-type haemocyanin. It has been shown (Hall et al, 1975) that L. stagnalis haemocyanin behaves in some ways as a fl-type haemocyanin.…”

“…It is not clear whether digestion had proceeded to completion. Gielens et al (1975) subjected their high-molecular-weight fraction to a further separation by ion-exchange chromatography on DEAE-cellulose and so obtained three components which they called TIA, TIB and TIC. By comparing the c.d.…”

Light-scattering investigation of the dissociation behavior of Lunatia heros and Littorina littorea hemocyanins

“…In both concentrated urea solutions and at high pH, the hemocyanins retain much of their secondary structure as suggested by their CD and absorbance spectra. The drop in molecular masses in 6.0 M GdmCl much below 4.5 X lo5 daltons seem to reveal some of the hidden breaks in the polypeptide chains of hemocyanin, which are known to be very susceptible to proteolysis (Gielens et al, 1975;Brouwer et al, 1978). The extrapolated values of the molecular masses close to 9 X lo6 daltons obtained with Lunatia hemocyanin are encouraging but require critical comments and further study.…”