Separation and absorption spectra of α- and β-haemocyanin of Helix pomatia

Heirwegh, K. P. M.; Borginon, H.; Lontie, René

doi:10.1016/0006-3002(61)90049-x

Cited by 132 publications

(36 citation statements)

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“…A different situation is encountered in Helix ponzatia where the existence of two distinct hemocyanin forms, termed a-hemocyanin and p-hemocyanin, has long been established (Heirwegh et al, 1961). This has prompted most researchers to work with either the one or the other purified form.…”

Section: Discussionmentioning

confidence: 99%

“…This has prompted most researchers to work with either the one or the other purified form. However, although ahemocyanin dissociates into subunits at pH 5.7 where P-hemocyanin is fully stable (Heirwegh et al, 1961 ; see also Gielens et al, 1987, for Helix aspersa), similarities to KLHl and KLH2 are limited since the two Helix hemocyanins are closely related immunologically (Preaux et al, 1981), contain eight functional domains each and show no multidecamer formation (Preaux et al, 1983;van Holde and Miller, 1995). Thus, it is unlikely that a-hemocyanin and P-hemocyanin of H. pomatia directly correspond to KLHI and KLH2.…”

Section: Discussionmentioning

confidence: 99%

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Mass Determination, Subunit Organization and Control of Oligomerization States of Keyhole Limpet Hemocyanin (KLH)

Söhngen¹,

Stahlmann²,

Harris³

et al. 1997

European Journal of Biochemistry

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Analytical dark-field scanning transmission electron microscopy (STEM) of freeze-dried unstained specimens of keyhole limpet hemocyanin (KLH ; from Megathura crenulata, a prosobranch gastropod) gave a molecular mass of 400 kDa for the subunit of KLHl and of 345 kDa for the subunit of KLH2, which confirms our published values from SDS/PAGE. Within the 400-kDa KLHl subunit we identified, by limited proteolysis, isolation of fragments and N-terminal sequencing, eight distinct 45 -60 kDa functional domains (termed l a through lh) and determined their sequential arrangement. The KLHl domains differ biochemically and immunologically from each other and from the previously characterized seven domains of KLH2 (termed 2a through 2g). Our partial amino acid sequences suggest that a domain, equivalent to the C-terminal domain lh, is missing in KLH2. This deficiency is believed to be genuine and not an artifact of the subunit preparation procedure, since STEM measurements of the native didecamers yielded a mass difference of about 800 kDa between KLHl and KLH2 (8.3 MDa versus 7.5 MDa), correlating with 20 copies of a functional l h domain. It was also shown that the KLHl didecamer can be rapidly split (minutes) into an almost homogeneous population of stable decamers by increasing the pH of the Triskaline stabilizing buffer (routinely pH 7.4), which contains 5 mM CaCl, and 5 mM MgCI,, to pH 8.5. Reformation of the didecamers occurred more slowly (days) upon dialysis against the pH 7.4 stabilizing buffer. Addition of 100 mM calcium and 100 mM magnesium ions to the pH 7.4 stabilizing buffer leads to the more rapid (overnight) formation of didecamers together with a significant number of previously unobserved KLHl multidecamers, which could be structurally distinguished from the established multidecamers of KLH2.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Mass Determination, Subunit Organization and Control of Oligomerization States of Keyhole Limpet Hemocyanin (KLH)

Söhngen¹,

Stahlmann²,

Harris³

et al. 1997

European Journal of Biochemistry

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“…In particular, the dispersion of the longitudinal (spin-lattice) relaxation rate 1/ Ti shows a monotonic decrease with increasing field, with an inflection point corresponding to a proton Larmor frequency which is inversely proportional to the orientational relaxation time of the protein. We have now compared the relaxation dispersion of solvent 1H, 2H, and 170 in aqueous solutions of lysozyme (molecular weight 14,700) and 'H and 2H in solutions of hemocyanin (molecular weight 9 X 106). The main experimental observation is that the dispersion of the relaxation rates of the three solvent nuclei in lysozyme solutions, normalized to their respective rates in pure water, is essentially the same.…”

mentioning

confidence: 99%

Protein-water interaction studied by solvent 1H, 2H, and 17O magnetic relaxation.

Koenig¹,

Hallenga²,

Shporer³

1975

Proc. Natl. Acad. Sci. U.S.A.

103

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Previous studies of the magnetic field de- Solvent proton relaxation in protein solutions can be accounted for reasonably well within the framework of a simple model of water exchange between the bulk solvent, in which the protons are assumed not to interact with the solute, and regions on or near the protein where the rotational motion of the protein molecules is conveyed to the water molecules. If it is assumed that the water molecules in the latter region attach rigidly to the protein, then one can readily estimate (1) the number n of such attachment sites to be about 3 for lysozyme and to increase at least as fast as the surface area for larger proteins (K. Hallenga and S. H. Koenig, to be published). If the attachment is assumed not rigid, the derived value of n is greater by a small factor, but is still-and this is the major unsettling feature of this simple two-site model-only a few percent of the number of water molecules in the first hydration layer (1). The nature of such special sites is difficult to conceive.From the data for proton relaxation, one can only set fairly wide limits on the value (or distribution of values) of TM, the mean lifetime of a water molecule on the protein and a quantity which is a feature of the model itself. The application of the model gives TM > TR 10-7 sec, and TM < Tipr -10-4 sec, the relaxation time of the protons when on the protein. In an attempt to get a better measurement of TM, as well as to test other predictions of the models, we measured and report here comparisons of the magnetic field dependence of the relaxation rate of 170 in solutions of protein in 17O-enriched water, and deuterons in 2H20 solutions, with the proton data. Since deuterons in water relax about 10 times faster than protons, and 170 nuclei about 100 times faster than deuterons, the upper limit on the model value for TM is drastically reduced and the range for TM is drastically narrowed. We have also made measurements on an extremely large protein-hemocyanin associated to a M.W. of 9 X 106-in order to increase the lower limit of TrM. The experiments were quite successful in that -TM was squeezed into nonexistence: i.e., the data reported below cannot be explained by a simple two-region model with a defined life-

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“…Helix ponzatia a-hemocyanin was isolated according to [22] and Panulims interruptus according to [23]. H. pomatia ol-hemocyanin was dialyzed, after regenerating any possible 'aged' sites with hydroxylamine [24],against 0.1 M Tris-HCl containing 10 mM CaCl,, pH 8.2.…”

Section: Methodsmentioning

confidence: 99%