Modelo estocástico para la simulación de estanques con lemnáceas

Contrary to earlier observations (Morgan, 1981; Guthans and Morgan, 1982) HRG does not appear to be a transporter for metal ions but there is inconsistent evidence for this. Analysis of hyperzincemic plasma showed that HRG concentrations were similar to that of normal plasma which is not in line with it being a major Zn2+ binder (Failla et al., 1982). Importantly however, its interactions with other binding partners are enhanced by Zn2+ but it is unclear how much Zn2+ would normally be

30 bound to HRG in vivo. Metal ion titrations and equilibrium dialysis have indicated that as many as 10 Zn2+ ions can bind to rabbit HRG (Morgan, 1981) and human HRG (Horne et al., 2001) with the His residues being involved in metal coordination (Morgan, 1981). Evidence for His involvement has been demonstrated by chemically modifying the His residues with diethyl pyrocarbonate (DEPC) which caused HRG to lose the ability to bind both Zn2+ (Morgan, 1981; Burch et al., 1987) and heparin (Borza and Morgan, 1998). At physiological pH the imidazole ring is partially deprotonated therefore it is readily available for metal binding. It is likely that Zn2+ binding modifies the protein structure of HRG in order to mediate interactions with other biomolecules (Jones et al., 2005b). The His residues provide a good “anchorage point” for metal ions, particularly in unstructured proteins where the residues are far more accessible (Rowinska-Zyrek et al., 2013). It is thought that the extended conformation of the HRR allows easier metal ion access to the imidazole rings of the His residues. It has also been demonstrated that the binding of Zn2+ to the HRR is cooperative, so that the binding of the first Zn2+ ion could make it more favourable for subsequent Zn2+ ions to bind (Morgan, 1981).

As shown in Figure 1.10 the HRR from different species are highly conserved. There are some slight differences, however, which could suggest that there are differences in Zn2+ binding affinity. For example, human HRG has 12 GHHPH repeats whereas rabbit HRG has 15, and overall a longer HRR, which indicates that the latter could have the

31 capacity to bind more metal ions. Moreover, the repeating unit GHHPH is predominant in bovine, human, mouse and rat HRG whereas GPPPH is more common in rabbit HRG.

Figure 1.10 Sequence alignment of the HRR of HRG from various mammalian species. Amino acids are coloured according to their chemical properties: hydrophobic (red), acidic (blue), basic (magenta), hydroxyl/sulfhydryl/amines (green). Symbols represent conservation of amino acids: fully conserved (*), conservation between groups with strongly similar properties (:) and conservation between groups of less similar properties (.).

In contrast, there is very little homology between the linker regions connecting the HRR and PRR thus highlighting the importance of these unusual domains with regards to the functionality of HRG (Hulett and Parish, 2002).

The binding affinity of HRG for metal ions has been addressed by a number of research groups as summarised in Table 1.4, These indicate that HRG binds Zn2+ with logK~ 5-6which suggests it is a weaker metal binder than HSA. Although the binding of Zn2+ and Cd2+ showed a similar binding affinity, Morgan (1981) demonstrated that Zn2+ is the strongest binder of the two as it was able to inhibit heme binding to a much greater extent than Cd2+. The general consensus is that affinity data are difficult to obtain for HRG and these values may be underestimated due to the HUMAN PLLPMSCSSCQHATFGTNGAQRHSHNNNSS---DLHPHKHHSHEQHPHGHHPHAHHPH 354 BOVINE LPFPPPGLRCPHPPFGTKGNHRP---PHDHSSDE--- 263 RABBIT PLSPPFRPRCRHRPFGTNETHRFPHHRISVNI-IHRPPPHGHHPHGPPPHGHHPHGPPPH 343 RAT PQLPPGYP----PHSGANRTHRPSYNHSCNEHPCHGHRPHGHHPHSHHPPGHHSHGHHPH 346 MOUSE PQMLPGHS----GPSGTNRSHRPPHNHSCNEHPCHGQHPHGHHPHGQHPHGHHPHGQHPH 346 *:: :* ** * .. HUMAN ---EHDTHRQHPHGHHPHGHHPHGHHPHGHHPHGHHPHCHDFQD 395 BOVINE ---HHPHGHHPHGHHPHGHHPHGHHPPDNDFYD 293 RABBIT GHPPHGPPPRHPPHGPPPHGHPPHGPPPHGHPPHGPPPHGHPPHGPPPHGHPPHGHGFHD 403 RAT G---HHPHSHHSHGHHPPGHHPHGHHPHGHHPHGHHPHGHHPHGHDFLD 392 MOUSE G---HHPHGQHPHGHHPHGQHPHGHHPHGHHPHGDHPHGHHPHGHDFLD 392 : *** **** *** **** * :.* *

32 fact that binding of Zn2+ and Cd2+ to the protein show cooperativity. Additionally, most work has been carried out using rabbit HRG, as the protein is more abundant in rabbit serum, but this has a longer HRR region compared to the human form.

Table 1.4 Stability constants for metal ion-HRG complexes

Metal ion pH of study Species logK Reference

Cu 7.4 Rabbit 6 Morgan, 1981 Cd 7.4 Rabbit 6a Morgan, 1981 Zn 7.4 Rabbit 6a Morgan, 1981 Zn 7.1 Rabbit 5 Guthans and Morgan,

1982

Zn 7.4 Rabbit 6-6.6 Burch and Morgan, 1985 Zn 7.4 Human 5.4 Horne et al., 2001

a

Values must be taken with caution as due to cooperative binding of these metal ions the apparent Kd was qualitatively determined from competition experiments. However,

these are still in good agreement with other estimations.