Báo cáo khoa học: Thermal stability of homologous functional units of Helix pomatia hemocyanin does not correlate with carbohydrate content potx

Thermal stability of homologous functional units of Helix pomatia hemocyanin does not correlate with carbohydrate content Betu ¨ l T. Yesilyurt 1 , Constant Gielens 1 and Filip Meersman 2 1 Division of Biochemistry, Molecular and Structural Biology, Department of Chemistry, Katholieke Universiteit Leuven, Belgium 2 Division of Molecular and Nanomaterials, Department of Chemistry, Katholieke Universiteit Leuven, Belgium Hemocyanins are complex respiratory molecules found in the hemolymph of many arthropods and molluscs [1,2]. The hemocyanin of the roman snail Helix pomatia consists of three components: two a-components (a D -HpH and a N -HpH) and a b-com- ponent (b-HpH). They all show the cylindrical quater- nary structure (diameter 35 nm, height 38 nm), typical of gastropodan hemocyanins, comprising 20 subunits with a molecular mass of approximately 450 kDa each [1]. The b-HpH is composed of only one type of subunit (b-subunit), whereas both a-HpHs are composed of two types (a and a¢ subunits) [3]. Owing to the subunit homogeneity, structural investigations on HpH have mainly been performed on the b-component. The subunits themselves are folded into eight globular functional units (FUs) designated a to h from the N-terminus on (Fig. 1). Above pH 8, b-HpH dissociates into dimers (M r 9 · 10 5 ) of subunits [4]. Limited trypsinolysis of these dimers yields the fragments HpH- abc and HpH-ef and the FUs HpH-d, HpH-g and HpH-h (occurring as dimers h 2 ) [5]. Further limited proteolysis of the Keywords disulfide bridge; FTIR spectroscopy; functional unit; glycosylation; hemocyanin Correspondence F. Meersman, Division of Molecular and Nanomaterials, Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, B-3001 Leuven, Belgium Fax: +32 16 327990 Tel: +32 16 327355 E-mail: filip.meersman@chem.kuleuven.be (Received 12 February 2008, revised 11 April 2008, accepted 15 May 2008) doi:10.1111/j.1742-4658.2008.06507.x The thermal stability of the eight functional units of b-hemocyanin of the gastropodan mollusc Helix pomatia was investigated by FTIR spectros- copy. Molluscan hemocyanin functional units have a molecular mass of approximately 50 kDa and generally contain three disulfide bridges: two in the mainly a-helical N-terminal domain and one in the C-terminal b-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. pomatia b-hemocyanin, designated HpH-a to HpH-h, differ considerably in their carbohydrate content (0–18 wt%). Most functional units are excep- tionally 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 confor- mational stability of the functional units. Sequence comparison of mollus- can 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. Abbreviations FU, functional unit; HpH, Helix pomatia hemocyanin; T m , melting temperature. FEBS Journal 275 (2008) 3625–3632 ª 2008 The Authors Journal compilation ª 2008 FEBS 3625 fragments Hp H-abc and HpH-ef with subtilisin allows each of the FUs to be isolated [6]. The FUs are disulfide bond containing entities with a molecular mass of approximately 50 kDa. The cry- stal structures are available for a cephalopod FU, OdH-g, from Octopus dofleini hemocyanin [7] and for a gastropod FU, RtH2-e, from Rapana thomasiana hemocyanin [8]. The polypeptide chain is folded into two distinct domains, a mainly helical N-terminal domain containing the active site and a C-terminal domain with b-sheet structure. The active site consists of six histidine residues coordinating two copper atoms. The molluscan hemocyanin FUs are very sim- ilar in sequence [9]. Hence, it is generally assumed that their tertiary structures are comparable [10]. Most hemocyanins are glycoproteins. The b-HpH contains 7% carbohydrate, with the individual FUs having a variable carbohydrate content (Table 1) [11,12]. The glycosylation sites were located in FUs HpH-d, HpH-e, HpH-g and HpH-h; FUs HpH-c and HpH-f are devoid of carbohydrate [11]. For FUs HpH-a and HpH-b, the glycan attachment sites are not yet known. The Asn387 residue at the carboxyl termi- nus, using the numbering of HpH-d [13], is the most conserved glycosylation site [12]. The carbohydrate moiety of molluscan hemocyanins has received particu- lar interest because of its possible role in the immuno- stimulatory properties of these proteins [14]. Recently, it was demonstrated that the glycan chains are involved in the antigenicity of R. thomasiana hemocya- nin [15]. In the present study, we investigated the ther- mal stability of the homologous FUs of b-hemocyanin of H. pomatia and considered the role of the degree of glycosylation and the secondary structure variability on the observed differences in stability. Results The thermal stability of the FUs was monitored by FTIR spectroscopy. In particular, the amide I¢ region is sensitive to the secondary structure of the protein [16]. This spectral region is therefore generally used to follow conformational changes as a function of temperature. At 25 °C, the amide I¢ region of all b-HpH FUs consists of a broad band. Deconvolution and fitting of the spectrum showed the presence of several bands at different wavenumbers. The positions of the compo- nent bands of the amide I¢ region for FU HpH-d are shown in Fig. 2. The structural features and the ther- mal behaviour of this FU is representative of all other FUs. Therefore, we limit our discussion to HpH-d. The peak positions and band assignments are summa- rized in Table 2. The band at 1610 cm )1 is generally assigned to the side chain contributions of the amino acids. After the deconvolution and fitting of the infra- red spectra of all FUs in the amide I¢ region, the per- centages of a-helix and b-sheet structures in the native proteins were calculated. The results of the fitting are in agreement with previous work that investigated the percentages of a-helix with CD (Table 3). Fig. 1. Cartoon of a b-HpH subunit and its eight FUs (a to h). The cleavage sites, on limited proteolysis, of trypsin (solid) in the sub- unit and of subtilisin (dashed) in fragments HpH-abc and HpH-ef are indicated by arrows. Table 1. Carbohydrate content and melting temperatures of b-HpH functional units (FUs). FU Carbohydrate content (wt%) T m (°C) HpH-a 7.4 a 76.9 ± 0.7 HpH-b 11.6 a 72.7 ± 0.2 HpH-c 0 a 64.4 ± 0.5 HpH-d 6.0 a 80.9 ± 0.5 HpH-e 6.0 b 78.4 ± 0.3 HpH-f 0 b 79.4 ± 0.9 HpH-g 17.6 a 83.3 ± 0.7 HpH-h 6.6 a 80 HpH-abc 6.3 a 77.8 ± 1.4 a Wood et al. [11]. b Gielens et al. [12]. Fig. 2. Secondary structure analysis of FU HpH-d from b-HpHat 25 °C. Curve-fitting of the Fourier self-deconvoluted amide I¢ band (1600–1700 cm )1 ). The Gaussian curves underneath the curve rep- resent the various contributions to the amide I¢ band. The peak assignment is given in Table 2. Thermal stability of hemocyanin functional units B. T. Yesilyurt et al. 3626 FEBS Journal 275 (2008) 3625–3632 ª 2008 The Authors Journal compilation ª 2008 FEBS Figure 3A shows the changes in the amide I¢ band of FU HpH-d upon heating. As the temperature increases, the intensity of the band at 1650 cm )1 , which is assigned to a-helix structure, gradually decreases and the formation of two new bands at 1618 and 1683 cm )1 is observed. The simultaneous appearance of these two bands is indicative of the for- mation of a b-sheet rich aggregate in which the strands are oriented in an antiparallel manner [17]. The unfolding process is irreversible. A white gel due to the aggregation could be observed by eye. Figure 3B compares the deconvoluted spectra at 25 °C and 90 °C. Apart from the IR aggregation bands at 1618 and 1683 cm )1 , a clear peak can still be observed at 1650 cm )1 with a shoulder at 1640 cm )1 at 90 °C, which correspond to peaks found at 25 °C. This suggests that only partial unfold- ing has taken place. In other words, some secondary structure persists, even at high temperatures. Note, however, that the ratio of the peaks at 1640 and 1650 cm )1 has changed at high temperature. Because the b-sheet structure absorbs in the region 1620– 1640 cm )1 region, it is most likely that any change here indicates a loss of b-sheet structure. If we plot the intensities at 1618 and 1647 cm )1 versus temperature, a sigmoid curve is found with a midpoint temperature (T m ) of approximately 81 °C (Fig. 4). The peak at 1647 cm )1 corresponds to the band maximum. It is assumed that this peak reflects mainly the changes in a-helix structure. Both graphs yield the same midpoint values, implying that the loss of a-helix structure is concomitant with the appearance of the aggregation bands. The midpoint temperatures for all FUs as well as for the HpH-abc fragment are listed in Table 1. In a recent study, the transition midpoints of the FUs HpH-d and HpH-g of b-HpH in glycine-NaOH buffer (pH 9.6) were determined by differential scanning calo- Table 2. Secondary structure assignment of the peaks in the amide I¢ region a . Wavenumber (cm )1 ) Secondary structure 1617 b Intermolecular b-sheet 1627 b-Sheet 1635 b-Sheet 1644 Disordered 1654 a-Helix 1662 Turn 1671 Turn 1684 b Intermolecular b-sheet a After Barth & Zscherp [16]. b Present in the aggregated state. Table 3. Secondary structure content of b-HpH functional units (FUs). Values (%) determined by curve-fitting of the amide I¢ band of the respective FUs at 25 °C. FU a-Helix b-Sheet FTIR CD a FTIR HpH-a 39.0 45 29.7 HpH-b 25.0 23 25.0 HpH-c 24.4 30 32.3 HpH-d 23.1 26 29.8 HpH-e 27.9 24 35.2 HpH-f 15.0 19 32.1 HpH-g 31.4 30 16.3 HpH-h 21.3 25 20.6 a CD spectra between 200 and 250 nm were obtained on a Cary CD 61 spectropolarimeter (Varian, Monrovia, CA, USA) in 25 m M borax-HCl (pH 8.2) at 20 °C. The spectra were analysed using a dataset of reference protein spectra [39] (M. Finotto, C. Gielens and G. Pre ´ aux, unpublished data). Fig. 3. Temperature dependence of the amide I¢ region of FU HpH-d of b-HpH. (A) Non-deconvoluted spectra are shown every 5 °Cin the range 25–90 °C. The arrows indicate the direction of the spec- tral changes with temperature. (B) Fourier-deconvoluted IR spec- trum of FU HpH-d in the amide I¢ region at 25 °C (solid line) and 90 °C (dotted line). B. T. Yesilyurt et al. Thermal stability of hemocyanin functional units FEBS Journal 275 (2008) 3625–3632 ª 2008 The Authors Journal compilation ª 2008 FEBS 3627 rimetry [18]. The respective melting points were 76 °C for HpH-d and 85 °C for HpH-g, which is in rather fair agreement with our findings, given the differences in experimental conditions. To obtain further insight into the unfolding process, we calculated the difference spectra as a function of temperature. These are obtained by subtracting the spectrum at 25 °C from the spectra at higher tempera- tures. The positive peaks indicate an intensity increase due to newly formed structures at that wavenumber. Conversely, negative peaks indicate an intensity decrease and the loss of native structure as a result of the temperature increase. The difference spectra of HpH-d (Fig. 5A) are dominated by two strong positive peaks at 1618 and 1683 cm )1 , indicative of the aggregation of the thermally denatured proteins. In addition, in the early stages of the heat denaturation process, a negative peak can be observed at 1632 cm )1 (Fig. 5B). This suggests at least part of the b-sheet structure unfolds prior to the unfolding of a-helix and the formation of aggregates. Note that the formation of aggregates cannot be fully excluded in the temperature range 25–60 °C because a small population of aggregate may go undetected. The early onset of protein aggregation is particularly apparent from the intensity changes at 1618 cm )1 in the case of FU HpH-h (Fig. 6). Above 55 °C, the intensity at 1618 cm )1 starts to increase, with a fur- ther increased slope in the range 75–85 °C. This made it impossible to fit the data to a single sigmoidal curve. Therefore, the T m was estimated visually, assuming the steepest part of the curve reflects the global unfolding of HpH-h. Discussion The thermal stability of the eight FUs of b-HpH was investigated. Six of these FUs were found to be highly temperature resistant with a T m close to 80 °C, whereas the two remaining FUs, HpH-c and, to a les- ser extent, HpH-b, are less stable. The reduced stability Fig. 5. Difference spectra for FU HpH-d. Difference spectra (DA) were obtained by subtracting the spectrum at 25 °C from all other spectra (A) in the temperature range 25–90 °C and (B) detail of the changes in the range 25–60 °C. Fig. 6. Intensity of the band at 1617.9 cm )1 versus temperature for FU HpH-h of b-HpH. Dots represent the experimental data and the full line is a guide to the eye. Fig. 4. Temperature dependence of intermolecular b-sheet and a-helix structures. Intensity of the bands at 1617.9 cm )1 (d) and 1646.9 cm )1 ( ) versus increasing temperature for FU HpH-d of b-HpH. These bands are assigned to intermolecular b-sheet and a-helix structures, respectively. Dots represent the experimental data, and the full lines are the fitted curves. Thermal stability of hemocyanin functional units B. T. Yesilyurt et al. 3628 FEBS Journal 275 (2008) 3625–3632 ª 2008 The Authors Journal compilation ª 2008 FEBS of HpH-c is particularly striking because it is destabi- lized by 15 °C compared to HpH-f, which also lacks carbohydrate groups. A possible explanation for the lower stabilities of HpH-b and HpH-c, discussed at length below, is that both FUs lack a third disulfide bridge in the C-terminal domain. Note, however, that the HpH-abc fragment, from which HpH-b and HpH-c are isolated, has a relatively high stability (Table 1), suggesting that the interactions within the fragment stabilize these FUs. The b-domain unfolds prior to the a-domain The observed unfolding behaviour of the FUs suggests that the C-terminal b-domain is less stable than the a-domain. This is not surprising because the a-domain contains the active site and the presence of the copper atoms will contribute to the stability of the protein. It has previously been shown for the subunit of b-HpH that the apo-form (copper-deprived) of the protein is destabilized by approximately 20 °C [19]. In addition, the a-domain also contains a cysteine-histidine thioe- ther bond and, for the known structures of OdH-g and RtH2-e [7,8], two out of three disulfide bridges, which will contribute to its stability [20]. The lower stability of the b-domain might explain the pronounced early onset of the aggregation of FU HpH-h which has, in comparison with the other FUs (HpH-a-g), an extra stretch of approximately 100 amino acids at the C-ter- minal end of the polypeptide chain [12]. This tail extension is generally found in gastropod hemocyanins [21], and has also been documented for the bivalve Nucula nucleus [20]. Note that, in the case of HpH-h, the C-terminal domain also lacks an attached glycan structure [12]. The aggregation propensity may be further enhanced by the fact that HpH-h is also resp- onsible for the dimerization of two subunits via nonco- valent interactions [11]. Thermal stability does not correlate with carbohydrate content Glycoproteins can be found in most organisms, includ- ing Archaea and bacteria [22]. The carbohydrate moi- ety plays an important role in protein trafficking and in molecular recognition phenomena. It also influences protein folding and, in some cases, glycosylation defects are associated with human diseases [22,23]. Moreover, it is commonly observed that the glycosy- lated protein has an increased thermal stability com- pared to the nonglycosylated form and that it is less susceptible to proteolysis [24–26]. The increased ther- mal stability, typically in the order of 4 °C [27,28], is generally explained by the fact that the carbohydrate moiety increases the chain stiffness near its attachment site. As a result, the entropy gain upon unfolding is reduced and the native-unfolded equilibrium is shifted slightly to the left [24–26]. Hence, we expected to find an increase in T m with increasing glycosylation con- tent, although it should be emphasized that we have investigated a series of homologous proteins rather than a single protein with or without glycans. To examine the effect of glycosylation on the ther- mal stability of homologous FUs, we plotted the T m values of the eight b- HpH FUs against their carbohy- drate content (Fig. 7). Although the FU with the high- est degree of glycosylation, HpH-g, has the highest melting temperature, there is no overall correlation between the T m and the carbohydrate content. It is particularly noteworthy that HpH-f, which lacks glycans, is more stable than the glycosylated FUs HpH-a, HpH-b and HpH-e. Different glycosylation sites and glycan structures can affect the stability in different ways, leading to the observed fluctuation of T m [23,25]. Moreover, as noted above, there is a strik- ing difference in stability between HpH-c and HpH-f, which are both devoid of attached glycans. Influence of the secondary structure variability on the thermal stability Molluscan hemocyanin FUs are known to have a large sequence homology. In the particular cases of HpH-d and HpH-g, for which the amino acid sequences are known, there is 56% sequence homology with 46% identity [13,29]. Taken together with the observation that the known crystal structures of FUs from two dif- ferent organisms are very similar [7,8], we conclude that the FUs of b-HpH are also structurally similar. However, despite the sequence homology, both FTIR Fig. 7. Midpoint temperatures (T m ) versus carbohydrate content of b-HpH FUs a to h. B. T. Yesilyurt et al. Thermal stability of hemocyanin functional units FEBS Journal 275 (2008) 3625–3632 ª 2008 The Authors Journal compilation ª 2008 FEBS 3629 and CD spectroscopy reveal a large variability in a-helix and b-sheet content of b-HpH FUs (Table 3). Nevertheless, the lack of any correlation between secondary structure and stability (data not shown) suggests that the conformational variability amongst the FUs is not a determining factor for the differences in their stabilities. Reduced stability of HpH-b and HpH-c The lower stability of FUs HpH-b and HpH-c is possi- bly explained by the absence of a disulfide bridge in the C-terminal domain. The stabilizing effect of the presence of disulfide bonds is well documented [30–32]. There is a rationale for our present hypothesis. Three disulfide bridges have been localized in b-HpH FUs HpH-d and HpH-g by protein chemical analysis [33] and in FUs OdH-g (from O. dofleini hemocyanin) and RtH2-e (from R. thomasiana hemocyanin) by X-ray crystallography [7,8]. Two of these disulfide bridges lie in the N-terminal core domain; the third one is situ- ated in the C-terminal domain. For all other molluscan hemocyanin FUs with known sequence, the disulfide bridges are assumed to be located at corresponding positions. However, sequence determination by cDNA analysis performed in our laboratory on the hemo- cyanin of the cephalopod Sepia officinalis has demon- strated that FUs SoH-b and SoH-c lack the third disulfide bridge near the carboxyl terminus (GenBank accession no. DQ388569 for subunit 1 and DQ388570 for subunit 2). The same absence of the third disulfide bridge has been observed for FUs OdH-b and OdH-c from O. dofleini Hc and for FUs NpH-b and NpH-c from Nautilus pompilius, suggesting this is a general feature of cephalopod hemocyanin [34,35]. Moreover, in the case of the gastropods Aplysia californica and Haliotis tuberculata,FUb lacks a third disulfide bridge, whereas, for the bivalve N. nucleus, a third disulfide bridge is missing in both FUs NnH-b and NnH-c of subunit 2 [20,21]. Given the sequence homo- logy between various mollusc species, it can be assumed that the third disulfide bond is also lacking in FUs HpH-b and HpH-c. In the case of R. thomasiana hemocyanin, it was found that a reduction of all three disulfide bridges resulted in a decrease of the T m by approximately 6 °C [30]. This would correspond to approximately 2 °C per disulfide bridge, assuming every disulfide bridge contributes equally. This would explain the differences in T m between HpH-b and, for example, HpH-a. In the case of HpH-c, other factors must be involved in addition to the absence of stabi- lizing glycan structures and a third disulfide bridge (see below). Molluscan hemocyanin has been shown to possess phenoloxidase activity [36], which can be increased by limited proteolysis [37]. Interestingly, we recently observed that the induced phenoloxidase activity is extremely high for HpH-c, whereas the induced activity of HpH-a and HpH-b, which together with HpH-c are isolated from the same HpH-abc fragment, is negligible (N. I. Siddiqui and C. Gielens, unpublished data). It is noteworthy that the thermal stability of HpH-a is com- parable to that of the HpH-abc fragment, and that, although HpH-b has a slightly lower stability, only HpH-c is significantly destabilized upon isolation (Table 1). It cannot be excluded that the purification procedure, which involves limited proteolysis, affects the structure and hence the stability of the other FUs as well. However, most FUs are found to be extremely thermally stable with an average T m of 80 °C, suggest- ing the purification procedure does not induce any sig- nificant conformational changes. Consistent with this view is the finding that the active sites of the fragments are well preserved [38], and that an analysis of the molar mass does not reveal any other fragments than the expected FUs [6]. Presumably the increased pheno- loxidase activity of HpH-c reflects a more dynamic structure, which could be correlated with a lower ther- mal stability. This is presently under investigation in our laboratory. In summary, an analysis of the thermal stabilities of the FUs of b-hemocyanin from H. pomatia indicates there is no linear correlation between the stability and the degree of glycosylation, nor with the secondary structure content. An intriguing finding is the lower stability of HpH-b and especially of HpH-c, which is attributed, at least in part, to the fact that these FUs have one disulfide bridge less compared to the other FUs. In future studies, the role of the disulfide bridges in determining the thermal stability of molluscan hemocyanin will be explored further. Experimental procedures Sample preparation All functional units (HpH-a-h) were obtained as described previously [6,37]. Briefly, a solution of didecameric mole- cules (M r 9 million) of b-HpH in phosphate buffer (pH 6.5) was dialysed against Tris–HCl (pH 8.2, I 50 mm), resulting in the dissociation of the didecamer into dimers of subunits. The solution was then subjected to limited prote- olysis with trypsin, yielding the fragments HpH-abc and HpH-ef and the FUs HpH-d, HpH -g and HpH-h (occurring as dimers h 2 ). The fragments were separated chromato- graphically and the FUs HpH-a, HpH-b and HpH-c, and Thermal stability of hemocyanin functional units B. T. Yesilyurt et al. 3630 FEBS Journal 275 (2008) 3625–3632 ª 2008 The Authors Journal compilation ª 2008 FEBS HpH-e and HpH-f, were isolated from HpH-abc and HpH-ef, respectively, by limited proteolysis using subtilisin (Fig. 1). The chromatographically separated FUs were concen- trated by ultrafiltration and dialysed against Milli-Q water (Millipore, Molsheim, France) to remove salt ions. Sub- sequently, the samples were lyophilized using a Savant Speedvac concentrator (Savant, Farmingdale, NY, USA). The dried protein was dissolved in deuterated Tris–DCl buffer (50 mm, pD 8.2) at approximately 50 mgÆmL )1 . The protein solution was stored overnight to ensure complete hydrogen-deuterium exchange of all solvent-exposed hydro- gens. To remove insoluble material, if any, the solution was centrifuged at 12 100 g for 3 min before use. FTIR spectroscopy Infrared spectra were recorded on a Bruker IFS66 FTIR spectrometer (Bruker, Ettlingen, Germany) equipped with a liquid nitrogen cooled mercury cadmium telluride detector at a nominal resolution of 2 cm )1 . Each spectrum is the result of the accumulation and averaging of 256 interfero- grams. The sample compartment was continuously purged with dry air to minimize the spectral contribution of atmo- spheric water. The heat unfolding was followed using a temperature cell with CaF 2 windows separated by a 50 lm teflon spacer. The cell was placed in a heating jacket controlled by a Graseby Specac Automatic Temperature Controller (Spelac Ltd, Orpington, UK). The temperature increment was 0.2 °CÆmin )1 . Buffer spectra as a function of temperature were recorded independently and subtracted from the solution spectra at the corresponding temperature. A baseline correction was performed in the amide I¢ region (1600–1700 cm )1 ) assuming a linear baseline. To enhance the component peaks contribut- ing to the amide I¢ band, the spectra were treated by Fourier self-deconvolution using the software provided by Bruker (OS ⁄ 2 version). The lineshape was assumed to be Lorentzian with a half-bandwidth of 21 cm )1 and an enhancement fac- tor k of 1.7 was used. Curve-fitting of the amide I¢ band was performed using grams ⁄ 32 (Thermo Galactic Inc., Salem, NH, USA) software after deconvolution of the spectrum using a c-factor of 11 and a 75% Bessel smoothing function. Acknowledgement F. Meersman is a Postdoctoral Fellow of the Research Foundation-Flanders (FWO-Vlaanderen). References 1 Pre ´ aux G & Gielens C (1984) Hemocyanins. In Copper Proteins and Copper Enzymes, Vol. II (Lontie R, ed.), pp. 159–205. CRC Press, Boca Raton, FL. 2 van Holde KE & Miller KI (1995) Hemocyanins. Adv Prot Chem 47, 1–81. 3 Lontie R (1983) Components, functional units, and active sites of Helix pomatia hemocyanin. 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Yesilyurt et al. 3632 FEBS Journal 275 (2008) 3625–3632 ª 2008 The Authors Journal compilation ª 2008 FEBS . Thermal stability of homologous functional units of Helix pomatia hemocyanin does not correlate with carbohydrate content Betu ¨ l T. Yesilyurt 1 , Constant. 2008) doi:10.1111/j.1742-4658.2008.06507.x The thermal stability of the eight functional units of b -hemocyanin of the gastropodan mollusc Helix pomatia was investigated by FTIR spectros- copy. Molluscan hemocyanin functional units have. the functional units of H. pomatia b -hemocyanin, designated HpH-a to HpH-h, differ considerably in their carbohydrate content (0–18 wt%). Most functional units are excep- tionally stable with