Thermal stability of homologous functional units of Helix pomatia hemocyanin does not correlate with carbohydrate content

Abstract

The thermal stability of the eight functional units of β‐hemocyanin of the gastropodan mollusc Helix pomatia was investigated by FTIR spectroscopy. Molluscan hemocyanin functional units have a molecular mass of approximately 50 kDa and generally contain three disulfide bridges: two in the mainly α‐helical N‐terminal domain and one in the C‐terminal β‐sheet domain. They show more than 50% sequence homology and it is assumed that they adopt a similar conformation. However, the functional units of H. pomatiaβ‐hemocyanin, designated HpH‐a to HpH‐h, differ considerably in their carbohydrate content (0–18 wt%). Most functional units are exceptionally stable with a melting temperature in the range 77–83 °C. Two functional units, HpH‐b and HpH‐c, however, have a reduced stability with melting temperature values of 73 °C and 64 °C, respectively. Although the most glycosylated functional unit (HpH‐g) has the highest temperature stability, there is no linear correlation between the degree of glycosylation of the functional units and the unfolding temperature. This is ascribed to variations in secondary structure as well as in glycan attachment sites. Moreover, the disulfide bonds might play an important role in the conformational stability of the functional units. Sequence comparison of molluscan hemocyanins suggests that the less stable functional units, HpH‐b and HpH‐c, similar to most of their paralogous counterparts, lack the disulfide bond in the C‐terminal domain.

Influence of limited proteolysis, detergent treatment and lyophilization on the phenoloxidase activity of Rapana thomasiana hemocyanin

“…From Fig. 7 it is clear that there is no significant effect of lyophilization on the secondary structure, which is very similar to that reported previously for H. pomatia Hc [23]. The absence of any secondary structure changes is corroborated by far-UV CD data where clear minima at 208 and 222 nm, typical of ␣-helix structure, can still be observed (Fig.…”

O-Linked Glycosylation Leads to Decreased Thermal Stability of Interferon Alpha 2b as Measured by Two Orthogonal Techniques

“…These researchers found that for this complex respiratory protein from the roman snail there was no linear correlation between the level of glycosylation and the unfolding temperature of its functional units, which share 50% sequence homology (24). Furthermore, Spiriti and coworkers studied the effect of O-linked glycosylation on a miniprotein analog of the macrophage-activating factor Gc-MAF (25).…”

Extraordinary stability of hemocyanins from L. polyphemus and E. californicum studied using infrared spectroscopy from 294 to 20 K

“…31,32 This band can be used to follow conformational changes induced by an external parameter, such as temperature. 33,34 Fig . 1 shows the comparison of the amide I region for both Hcs at 294 K and 20 K and for different pH values (pH 7, 7.5/7.8 and 8.5).…”