In the case presented here, data are presented investigating the binding of HPRG
ID: 52213 • Letter: I
Question
In the case presented here, data are presented investigating the binding of HPRG to heparin. Heparin is an anionic polysaccharide (Figure 2.2) that is one of the major components of the ground substance that forms the matrix of the extracellular spaces of the connective tissue in blood vessel walls. Based on the data below, the original investigators proposed a model that describes how binding of HPRG to heparin may allow HPRG to regulate local blood pH. The local pH in blood may drop a half a pH unit during lactic acidosis or even a full pH unit in hypoxia or ischemia Questions 1. Binding studies were carried out in which heparin was 700 600 500 400 300 200 100 immobilized on the surface of cuvettes. The phH dependence of HPRG binding to heparin was investigated and the results are shown in Figure 2.1 HPRG Using Figure 2.1, describe the pH dependence of HPRG binding to heparin. Provide structural reasons for this dependence in your explanation. (NOTE: The structure of heparin is shown in Figure 2.2) a. DEPC-HPRG 5.2 5.7 6.2 6.7 7.2 pH Figure 2.1: The pH-dependence of HPRG binding to unmodified heparin and heparin modified with DEPC b. Figure 2.1 can also be interpreted as a titration curve for the His residues in HPRG-what is the pKR for these residues?) OH The same binding studies were carried out in which HPRG was reacted with heparin modified with diethylpyrocarbonate (DEPC), a compound that specifically reacts with histidine residues (Figure 2.1). Explain these results. The Figure 2.2: Repeating disaccharide unit of heparin reaction of DEPC and HPRG is shown in Figure 2.3 c. 0-Explanation / Answer
1. a. Heparin is a negative charged compound whereas HPRG has a high affinity towards it due to the presence of histidine in it. The histidine residue is become protonated and becomes positively charged when the pH drops below the pKa of histidine which is 6. Hence the positively charged HPRG binds to the negatively charged heparin. Hence the binding is more at low pH but as the pH increased the histidine becomes unprotonated and didn't able to bind to effectively with heparin.
c. When HPRG treated with DEPC it reacts with the imidazole ring and modifies the structure of histidine. This makes the DEPC-HPRG unable to bind to heparin due to prevention of protonation. Hence the graph shows zero binding.
2. The binding of HPRG to heparin was studied at pH 7.3 with metal ions such as zinc and copper. It may be found that at high pH the binding of HPRG to heparin was low. The presence of metal ions such as zinc and copper at concentration 1.0M increased the binding. Although copper increase the binding compared to zinc it is lower. In lower concentration of 0.1M the binding was same for copper and zinc.
3. The binding of HPRG with heparin is decreasing with increasing pH in the absence of zinc ions. But when the zinc is added with increasing concentration the binding is above than without zinc ions in a significant manner.
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