Journal of Biomedical Technology and Research

The effects of metal ions on heparin/heparin sulfate-protein interactions

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Published Date: May 19, 2014

The effects of metal ions on heparin/heparin sulfate-protein interactions

Fuming Zhang1* , Xinle Liang2, Julie M Beaudet3, Yujin Lee3 and Robert J Linhardt1,3,4

1Department of Chemical and Biological Engineering

2Department of Bioengineering, School of Food Science and Biotechnology, Zhejiang Gongshang, University, Hangzhou 310025, China

3Department of Biology

4 Departments of Chemistry and Chemical Biology and Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.

*Corresponding author: Fuming Zhang, Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA

Citation: Fuming Zhang, Xinle Liang, Julie M Beaudet, Yujin Lee, Robert J Linhardt (2014) The effects of metal ions on heparin/heparin sulfate-protein interactions. J Biom Tech Res 1(1): 6000101. Doi:



Heparin/heparin sulfate (HS) interacts with a number of proteins thereby playing an essential role in the regulation of many physiological processes. The understanding of heparin/HS-protein interactions at the molecular level is of fundamental importance to biology and will aid in the development of highly specific glycan-based therapeutic agents. The heparin-binding proteins (HBPs) interact with sulfated domains of heparin/HS chains primarily through ionic attraction between negatively charged groups in HS/heparin chains and basic amino acid residues within the protein. Reports in literature have been shown that heparin molecules have a high affinity for a wide range of metal ions. In the present study, we used surface plasmon resonance (SPR) to study the effects of metal ions (underphysiological and nonphysiological concentrations)on heparin/HS-protein interactions.The results showed that under non-physiological of metal ion concentration, different metal ions showed different effects on heparinbinding to fibroblast growth factor-1 (FGF1) and interleakin-7 (IL7). While the effects of individualmetal ion at physiological concentrationshad little impact on protein binding, the mixed metal ions reduced the FGF1/heparin or IL7/heparin binding affinity, changing its binding profile.

Keywords: Heparin, Potein, Metalion, Interaction, Surface Plasmon Resonance.

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SPR, surface plasmon resonance; GAG, glycosaminoglycan; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HS, heparan sulfate; RU, resonance unit,FGF1, Fibroblast growth factor 1; IL7, Human interleukin 7.

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Heparin/heparan sulfate (HS) glycosaminoglycans (GAGs) are anionic and often highly sulfated, polydisperse linear polysaccharides. GAGs are ubiquitous molecules exhibiting a wide range of biological functions by interaction with various growth and differentiation factors and morphogens, extracellular matrix components, protease inhibitors, protease, lipoprotein lipase, and various pathogens [1-4]. Interactions between heparin/HS and proteins ediate diverse patho-physiological processes including: blood coagulation, cell growth and differentiation, host defense and viral infection, lipid transport and metabolism, cell-tocell and cell-to-matrix signaling, inflammation, angiogenesis and cancer [4-6]. Thus, an understanding of heparin/ HS-protein interactions at the molecular level is of fundamental importance to biology and should aid in the development of highly specific glycan-based therapeutic agents [3,5]. Metals play crucial roles in biological processes which are involved in cellular and subcellular functions [7]. For instance, the divalent magnesium and calcium ions play important regulatory roles in cells. Lack of body iron is common in cancer patients and it is associated with complications in surgery and in animal experiments. Metal ions play essential roles in about one third of enzyme interactions [8]. These ions can modify electron flow in a substrate or enzyme, thus effectively controlling an enzyme-catalyzed reaction. They can serve to bind and orient substrate with respect to functional groups in the active site of the enzyme [9]. Copper is recognized as an essential metalloelement and is primarily associated with copper-dependent cellular enzymes. Metal ions function in numerous metalloenzymes, are incorporated into pharmaceuticals and used as inorganic drugs for many diseases [7,10]. The heparin-binding proteins (HBPs) interact with sulfated domains of HS/heparin chains by ionic attraction between negatively charged groups in HS/heparin chains and basic amino acid residues in the protein. Previous study has shown that heparin molecules have a high affinity for a wide range of metal ions [11-19], which suggests the presence of metals may play a significant role in heparin/HS-protein interactions. For example, divalent cations play an important role in regulating the anti-Factor Xa activity of heparin [20]. It has also reported that divalent cations and heparin/heparan sulfate cooperate to control assembly and activity of the fibroblast growth factor [21]. Unfortunately, the effects of metal ions on protein-heparin/HS complexes and their biological activities are largely unknown. Thus, we undertook this study to evaluate the impact of metal ions protein-heparin/HS interaction. The present study uses surface plasmon resonance (SPR) spectroscopy to evaluate the effect of common metal ions on heparin/HS interactions
with fibroblast growth factor-1 (FGF1) and interleukin-7 (IL7). 

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Materials and Methods

Materials. Porcine intestinal heparin (16 kDa) and porcine intestinal heparan sulfate (12 kDa) were obtained fromCelsus Laboratories (Cincinnati, OH). Sensor SA chips were from GE Healthcare (Uppsala, Sweden). Fibroblast growth factor 1 (FGF1) was a gift from Amgen (Thousands Oaks, CA). Human interleukin 7 (IL7) was provided by Dr. Walsh (Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute). SPR measurements were performed on a BIAcore 3000 (GE Healthcare, Uppsala, Sweden) operated using BIAcore 3000 control and BIAevaluation software (version 4.0.1).

 Preparation of heparin biochip: Biotinylated heparin or HS was prepared by reaction of sulfo-N-hydroxysuccinimide long-chain biotin (Piece, Rockford, IL) with free amino groups of unsubstituted glucosamine residues in the polysaccharide chain following a published procedure [22]. Biotinylated heparin was immobilized to streptavidin chip based on the manufacturer’s protocol. In brief, 20-μl solution of the heparin-biotin conjugate (0.1 mg/ml) in HBS-EP running buffer was injected over flow cell 2 of the streptavidin chip at a flow rate of 10 μl/min. The successful immobilization of heparin was confirmed by the observation of a ~250 resonance unit (RU) increase in the sensor chip. The control flow cell was prepared by 1 min injection with saturated biotin.

Measurement of the effects of metal ions on the interaction between heparin/HS and protein (FGF1 or IL-7) Using SPR. The protein samples were diluted in HBS-P buffer (0.01 M HEPES, 0.15 M NaCl, 0.005% surfactant P20, pH 7.4). Different dilutions of protein samples with or without addition of metal ions were injected at a flow rate of 30 μl/min. At the end of the sample injection, the same buffer was flowed over the sensor surface to facilitate dissociation. After a 3 min dissociation time, the sensor surface was regenerated by injecting 30 μl of 2 M NaCl to obtain a regenerated surface. The response was monitored as a function of time (sensorgram) at 25 °C.

The additions of metal ions to theheparin/HS and protein binding measurement were in three categories: 1) addition of CaCl2, ZnCl2, FeCl3, MgCl2, and KCl in concentration of 0, 10, 100 and 1000 μM, respectively; 2) addition of metal ions in physiological lower/upper limit concentrations (Table 1); 3) the addition of mixed metals ions with Mg2+ (50μM), Zn2+(15μM), Fe3+ (20μM), K+(2000μM), Ca2+ (1150μM ), and Cu2+ (15μM)in physiological concentrations.

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Results and Discussion 


The effects of metal ions on heparin/HS-protein interactions in non-physiological concentrations

The first set of SPR measurements on heparin/HS-protein interaction were conducted through the addition of CaCl2, ZnCl2, FeCl3, MgCl2, and KCl at concentration of 0, 10, 100 and 1000 μM, respectively. FGF1 which is well known as a heparin-binding protein (HBP), was used in this initial experiment.

The results (Figures 1 to 3) showed that at non-physiological concentrations, different metal ions showed different effects on the heparin/HS-proteinbinding. The metal ions showed a greater effect on the HS-FGF1 interaction than on the heparin-FGF1 interaction and most of the effects of most of the metal ions were concentration dependent.FGF1 binding to heparin/HS was reduced with addition of Ca2+or Mg2+at 10 μM and the effects were decreased at 100 and 1000 μM concentrations of Ca2+or Mg2+. FGF1 binding to heparin/HS was unaffected with addition of 10 μM Zn2+butbinding was dramatically reduced atZn2+concentrations of 100 and 1000 μM. FGF1 binding to heparin/HS was reduced at 10 μMFe3+and further decreased at 100 μMFe3+ and no binding was detected at 1000 μMFe3+. FGF1 to heparin/HS was greatly reduced at all concentrations of K+, ranging from 10 to 1000 μM. Some studies have reported that physiological metal ions such as sodium, calcium, and magnesium bind to heparin based on the polyelectrolyte theory [23-26].


Figure 1. SPR sensorgrams of heparin-FGF1 interaction with the addition of metals ions.FGF1 concentration was 500 nM. A:heparin- FGF1 interaction with the addition of CaCl2 (0, 10, 100 and 1000 μM); B: heparin-FGF1 interaction with the addition of ZnCl2 (0, 10, 100 and 1000 μM); C: heparin-FGF1 interaction with the addition of FeCl3 (0, 10, 100 and 1000 μM); D: heparin-FGF1 interaction with the addition of MgCl2 (0, 10, 100 and 1000 μM); E:heparin-FGF1 interaction with the addition of KCl (0, 10, 100 and 1000 μM).


Figure 2. SPR sensorgrams of HS-FGF1 interaction with the addition of metals ions. FGF1 concentration was 500 nM. A: HS-FGF1 interaction with the addition of CaCl2 (0, 10, 100 and 1000 μM); B: HS-FGF1 interaction with the addition of ZnCl2 (0, 10, 100 and 1000 μM); C: HS-FGF1 interaction with the addition of FeCl3 (0, 10, 100 and 1000 μM); D: HS-FGF1 interaction with the addition of MgCl2 (0, 10, 100 and 1000 μM); E:HS-FGF1 interaction with the addition of KCl (0, 10, 100 and 1000 μM).


Figure 3. A: Normalized FGF1 binding to heparin in the presence of different concentration of metal ions.B: Normalized FGF1 binding to HSin the presence of different concentration ofmetal ions.


Using atomic absorption and spectrophotometry, it was reported the overall trend for heparin–metal affinity to be Mn2+>Cu2+>Ca2+>Zn2+>Co2+>Na+>Mg2+>Fe3+>Ni2+>Al3+>Sr2+. There is evidence that divalent metal ions (Ca2+, Cu2+, and Zn2+) are necessary in many protein-heparin interactions thus influencing the affinity, specificity and stability of these complexes [27-28]. A previous study by our group showed the conformational changes induced by calcium ions are necessary for the interaction between heparin and annexin V [25].

The effects of metal ions on heparin/HS-protein interactions in physiological concentrations

Next, heparin/HS FGF1 and heparin/HS-IL7 interactions were studied with the addition of metal ions at the physiological lower/upper limit concentrations (Table 1) [29]. The results (Figures 4 and 5) showed effects, of mostindividual metal ions atphysiological lower/upper limit concentrations, on these interactions were minimal. One exception was the effect of Cu2+on the interaction of heparin/HS with FGF1 (Figure 4). A second exception was the effect of Fe3+, at its upper limit concentration, on the interaction of heparin/HS with IL7 (Figure 5) obviously reduced with the addition of Fe3+ (Figure 5). We previously reported the formation of a Cu2+-heparin complex gave extremely sensitive detection of heparin, permitting the analysis of as low as 10 ng with capillary electrophoresis [30]. Copper, along with FGF, [31] plays an important role in promoting physiological and malignant angiogenesis,the formation of new blood vessels by a tumor, enabling tumor growth, invasion, and metastasis [22]. It also has been reported that the heparin-copper complex is angiogenicin vivo and stimulates migration of capillary endothelium in vitro [32].

Figure 4.A: FGF1 binding (RU) to heparin in the presence of physiological lower/upper limit concentrations of metal ions. FGF1 concentration was 500 nM; B:FGF1 binding (RU) to HSin the presence of physiological lower limit/upper limit concentrations of metal ions. FGF1 concentration was 500 nM.


Figure 5. A: IL7 binding (RU) to heparin in the presence of physiological lower/upper limit concentrations of metal ions. IL7 concentration was 500 nM; B: IL7 binding (RU) to HSin the presence of physiological lower /upper limit concentrations of metal ions.IL7 concentration was 500 nM.


The effects of mixed metal ions on heparin-protein interactions. Finally, we measured the effect of physical ogical concentrations of mixed metals ions in physiological concentrations, i.e. Mg2+ (50μM), Zn2+(15μM), Fe3+ (20μM), K+(2000μM), Ca2+ (1150μM ), and Cu2+ (15μM), on the heparin-FGF1 interaction. Sensorgrams ofheparin and FGF1 interactions are shown in Figure 6. The kinetic parameters (Table 2) of  FGF1/heparin interactionswere obtained by fitting thesensorgrams with a Langmuir 1:1 binding model. The SPR data showed different FGF1/heparinbinding profiles (Figure 6 A, and B) with and without the added mixed metals ions. Without the addition of mixed metals ions (control),the KD for FGF1/heparin interaction was 22 nM, with the addition of mixed metals ions,the KD for FGF1/heparin interaction was 350 nM. SPR analysis also showed different binding kinetics for FGF1/heparin interactionsin the absence and presence of mixed metals ions. Without the added mixed metals ions, FGF1/heparin interaction exhibitedaka=4.5 × 105(1/Ms), and a kd = 0.01 (1/s), while FGF1/heparin interaction exhibitedaka=1.6 ×104 (1/Ms) and a kd = 5.7 ×10-3 (1/s) the presence of mixed metals ions.


Figure 6. SPR sensorgrams of heparin-FGF1 or IL7 interaction with the addition of mixed metals ions in physiological concentrations. A: SPR sensorgrams of heparin-FGF1 interaction without addition of mixed metals ions;B: SPR sensorgrams of heparin-FGF1 interaction with addition of mixed metals ions; Concentrations of FGF1 (from top to bottom): 500, 250, 125 and 63 nM, respectively. C: SPR sensorgrams of heparin-IL7 interaction without addition of mixed metals ions; D: SPR sensorgrams of heparin-IL7 interaction with addition of mixed metals ions; Concentrations of IL7 (from top to bottom): 500, 250, 125 and 63 nM, respectively. The black curves are the fitting curves using models from BIAevaluate 4.0.1.


In conclusion, the results of this study clearly show different metal ions can have different effects on the heparin/HSproteinbindingatnon- physiological concentrations. Metal ions in the range of physiological concentrationswith few exceptions generally show little impact on heparin/HS-protein interactions. However, mixed metal ionscan alter binding affinity in the case of FGF1/heparin or IL7/heparinbinding. This study provides useful information for the formulation of heparin/HS-based agent to promote or block biological processes with heparin-protein interactions.

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This work was supported by grants from the National Institutes of Health in the form of GM-38060 to R.J.L.

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Copyright: © 2014 Fuming Zhang, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.