Estimation of the apparent binding constant for Zn-HRGP330 Zincon was used in a competition assay to determine an apparent binding

constant (Kapp) for HRGP330. Complexation of Zincon with Zn2+ produces

a 1:1 complex with a blue colour and a characteristic absorbance at 620 nm. This absorbance can be exploited to assess the binding of Zn2+ to peptides and proteins (Shaw et al., 1990; Mekmouche et al., 2005; Armas

et al., 2006; Talmard et al., 2007).

Titrating increasing amounts of HRGP330 into the Zn2+-Zincon complex produced a decrease in the absorbance at 620 nm (Figure 6.9). Approximately 4.8 µM HRGP330 was required to reduce the absorbance at 620 nm by 50% which indicates that half of the Zn2+ originally bound to Zincon had been transferred to HRGP330. A plateau in the absorbance is reached relatively quickly which demonstrates that Zn2+ exchange between HRGP330 and Zincon is rapid. The results indicate that the

163 binding constants are globally similar but HRGP330 is a slightly stronger Zn2+ binder than Zincon. The Kapp for Zincon has previously been

determined to be 7.9 x 104 M-1 at pH 7.4 (Shaw et al., 1990) and more recently a slightly different value of 8.5 x 104 M-1 was found by Mekmouche et al. (2006) under 50 mM HEPES, 100 mM NaCl at pH 7.4. Using this value, Equation 3 in the experimental methods can be used which results in a Kapp for HRGP330 of 2.7 x 105 M-1

Figure 6.9 Estimation of the apparent binding constant of Zn2+ and HRGP330 using competition with Zincon. HRGP330 was titrated into a solution of 10 µM Zincon and 5 µM ZnCl2 in 50 mM HEPES, 100 mM NaCl, pH 7.4. 100% relative absorbance

corresponds to the maximum absorbance observed at 620 nm at t=0 and t=∞. The inset shows the structure of Zincon.

As HRGP330 is able to obtain Zn2+ from the 1:1 Zn2+-Zincon complex this would suggest it is also able to rapidly acquire Zn2+ from other available donors, including HSA under appropriate conditions. This result yields an apparent logK of 5.43 which is in the same range as the values published

164 for full length HRG (Table 1.4) therefore it would appear that HRGP330 has a similar binding affinity to the intact protein. Differences will be due to the different buffer conditions and ionic strengths used. Recently, our laboratory has determined an apparent logK of 5.67 for Zn2+ binding to BSA in 50 mM Tris, 50 mM NaCl at pH 7.2 (Lu et al., 2012b). Therefore, although BSA or HSA would bind Zn2+ with greater affinity than HRG, the high number of potential binding sites may mean the binding capacity of HRG is underestimated. It would be invaluable to assess the binding affinity of HSA and human HRG under the same conditions by a method such as ITC although there may be difficulties in comparing a cooperative binder (HRG) to a non-cooperative binder (HSA).

6.2.7 Cu2+ transfer between Gly-Gly-His and HRGP330

The major Cu2+ binding site on HSA is the N-terminal ATCUN-motif. Gly- Gly-His is a peptide mimic of this site that has been previously studied as a model for the N-terminal site (Figure 6.10). The apparent stability constant for the Cu2+-peptide complex has been shown to be logK = 16 compared to 16.2 for Cu2+-HSA (Lau et al., 1974). Although most of the ligands that interact with HRG are thought to be mediated by Zn2+ binding to the HRR, these associations could also be influenced by the concentration of Cu2+. Borza and Morgan (1998) confirmed that the only other transition metal apart from Zn2+ to be effective at promoting heparin binding to HRG at physiological pH was Cu2+. Binding of Cu2+ to rabbit HRG has also been shown to be stronger than Zn2+ (Morgan, 1981). In fact, analysis of serum indicates that while the concentration of Zn2+ is

165 reduced in human cancers (Kuo et al., 2002), plasma Cu2+ levels are actually increased (Coates et al., 1989). Therefore, it must be acknowledged that Cu2+ could potentially be biologically relevant for HRG although the question still remains as to whether it contributes significantly to anti-angiogenic activity.

ESI-MS experiments of Gly-Gly-His showed binding of 1 equivalent of Cu2+ which is consistent with the literature. Cu2+ distribution between Gly- Gly-His and HRGP330 was also studied using this technique. First the Cu2+-Gly-Gly-His complex was formed which can be observed at 331 m/z

in spectra A and C in Figure 6.11.

Figure 6.10 Structure of Gly-Gly-His. The peptide mimic for the N terminus of HSA.

This was reacted with 1 or 0.2 mol. equiv. of HRGP330, shown in Figure 6.11 B and D respectively. With addition of 1 mol. equiv. of HRGP330, the intensity of ESI-MS peaks corresponding to the Cu2+-Gly-Gly-His species reduced considerably to 10% relative intensity from 69% and the peak at 270 m/z, corresponding to Gly-Gly-His without a metal ion, became the most abundant species (Figure 6.11 B). This showed that

H3N+ N H N N H N H O O O O– H3N+ N H N N H N H O O O O–

166

Figure 6.11 Effect of HRGP330 on Cu2+ binding to Gly-Gly-His. Cu2+ was allowed to react with Gly-Gly-His producing the spectra in A and C. HRGP330 was added at concentrations of 1 mol. equiv. (B) and 0.2 mol. equiv. (D). Samples were prepared in 10 mM ammonium acetate at pH 7.4 and analysed on a maXis-UHR-TOFinstrument.

Cu2+ transfer had occurred to HRGP330. A similar approach was to add 1 mol. equiv. of Cu2+ to a 1:1 HRGP330:Gly-Gly-His: the same result was achieved as the metal ion preferentially bound to HRGP330 over Gly-Gly- His. As HRGP330 was clearly shown to be able to bind up to 5 metal ions in Chapter 5, this reaction was repeated with 0.2 mol. equiv. HRGP330. This means that the potential binding sites between the two peptides are approximately equal. At this concentration there was a significant

167 increase in the 270 m/z peak to the point where a 50:50 ratio of Gly-Gly- His and Cu2+-Gly-Gly-His was observed. This experiment indicated that not all of the Cu2+ had been transferred to HRGP330. At this point it can be assumed that globally the binding affinities are roughly similar as approximately half of Cu2+ is bound to each peptide. This result is surprising as Gly-Gly-His binds to Cu2+ with a much stronger affinity than proposed for HRG: logK = 16 for Cu2+-Gly-Gly-His (Lau et al., 1974) compared to ~6.70 for Cu2+-HRG (Morgan, 1981). Therefore, it may be that case that the large number of potential binding sites makes Cu2+ binding to HRGP330 more favourable. These results indicate that one route through which HRG may obtain Cu2+ in plasma could be through transfer from HSA. Furthermore, as Cu2+ binds to HRG more strongly than Zn2+ then it would be feasible that Cu2+ would sufficiently block metal binding sites and this might have consequences for the Zn2+-dependent activities of HRG.

6.2.8 Implications of Zn2+ transfer to HRG: effect of Zn2+ on HRGP330