The Binding of Lipids to Bovine Serum Albumin - The University of ...

of LiThe BindinBovine Serum AlbuminWritten by: Amber BrownUniversity of MemphisChemistry DepartmentJune 2009Amber BDaniel Baker7-,q- *AQDate

Introduction:BSA (bovine serum albumin) is a common lipid found in the blood stream to keepthings such as blood cells from sticking together which could form dangerous blood clots.BSA also transports fatty acids and other lipids. Although binding BSA to fatty acids hasbeen done before 1-4 , it has not been done by using an (ITC) Isothermal TitrationCalorimeter. The ITC measures heat change when different lipids bind to the protein.But first, I had to convert Kd values that I got from previous research papers into Kvalues to get the target concentrations for these experiments. Kd values are numbers thatcome from finding the concentration of a lipid. Scientists such as Inje N. Bojesen andEijil Bojesen 1,4 have come up with these Kd values that I have converted into K.Planning target concentrations:K (M) are the concentrations of the lipids in molar which were used to plan thelipid and protein concentrations that could be used to study lipid:protein interactions byisothermal titration calorimetry (ITC) using this equation:c=KM tot nI used the Kd values to represent (K), (n) was set equaled to 3 because that’s the reportedbinding stoichiometry, and ‘c’ varied 5 . We used a range of ‘c’ values between 5 and 500because it was recommended in the ITC manual as a range giving a binding isotherm thatcould be used to obtain K, n and ∆H values. After figuring out M (the concentration)when (c) =5 or (c) =500, we came up with a BSA/HSA (Human Serum Albumin)concentration that was closest to M when (c) =500. For example, the BSA concentration2

needed with palmitate to give (c) =500 is M=5.67E-6 M 1 . So my BSA concentrationwould be 1.00E-6 M 1 . After getting my BSA concentrations, I multiplied them by 20 togive the lipid concentration needed to produce a binding isotherm within 29 injections of10μl each.TABLE 1: Preliminary target concentrations for lipid: protein binding experiments.Lipids Protein K(M) n M(c=5) M(c=500)BSA/HSAcon.Lipidcon. MPalmitate 1 BSA 2.94E+07 3 5.67E-08 5.67E-06 1.00E-06 2.00E-05Palmitate 1 BSA 6.36E+07 3 2.62E-08 2.62E-06Arachidonate 4 BSA 6.41E+07 3 2.60E-08 2.60E-06 1.00E-06 2.00E-05Anandamide 4 BSA 1.46E+08 3 1.15E-08 1.15E-06 1.00E-06 2.00E-05Anandamide 4 BSA 2.86E+07 3 5.82E-08 5.82E-06Anandamide 4 BSA 1.82E+07 3 9.15E-08 9.15E-06Anandamide 4 BSA 3.57E+07 3 4.67E-08 4.67E-06Oleic Acid 2 BSA 7.41E+08 3 2.25E-09 2.25E-07 1.00E-08 2.00E-07Oleic Acid 2 BSA 9.09E+09 3 1.83E-10 1.83E-08Oleic Acid 2 BSA 7.14E+08 3 2.33E-09 2.33E-07Oleate 2 BSA 3.46E+08 3 4.82E-09 4.82E-07Laurate 3 HAS 8.26E+06 3 2.02E-07 2.02E-05 1.00E-05 2.00E-04Myristate 3 HAS 3.10E+07 3 5.38E-08 5.38E-06 1.00E-06 2.00E-05Converting (Kd) values into the (K) values shown in Table 1 was done by taking thereciprocal. Table 1 shows how target protein: lipid concentrations were selected on thebasis of c values, which vary between 5 and 500 using the converted values.Making the protein solutions:I picked laurate as the first lipid I wanted to bind to BSA. The targetconcentration of laurate was 2.00E-4 M and the target concentration of BSA was 1.00E-5M (Table 1). When making my BSA stock solution, I measured out 67 mg of BSA andadded it to 10 ml of 1% DMSO (dimethyl sulfoxide) and PBS (phosphate buffer saline)which was my stock solution, to give a concentration of 0.1 mM. The DMSO in PBS3

stock was made by adding 2500 μl of DMSO into 247.5 ml of PBS. After making myBSA stock solution, I took 1000 μl of BSA in DMSO/PBS and 3000μl of 1%DMSO/PBSto get a concentration 0.025 mM for my protein solution. This solution went into thesample cell.Making the lipid solution:My lipid solution was lauric acid (laurate). When making the lauric acid solution,I measured out 100mg of lauric acid and mixed it with 10ml of DMSO to give aconcentration of 50mM. After making sure that the lauric acid was completely dissolvedin the DMSO, we diluted it 1:100 by taking 100μl of lauric acid in DMSO and put it in9900 μl of DMSO/PBS to get a concentration of 0.5 mM. This solution went into thesyringe, and degassed MilliQ water was placed in the reference cell.The Titration:Before I started the titration, I always checked to make sure that the instrumentwas cleaned because if not, then I would get very disturbing results at the end of thetitration. If the instrument wasn’t cleaned, then I would fill it with a cleaning solutionand let it sit for 15 to 20 minutes. After rinsing out the instrument numerous of times, Iwould start setting up for the titration. Once I knew what solution went into the cell(BSA in 1%DMSO/PBS) and what solution went into the syringe (lauric acid in1%DMSO/PBS), I degassed both solutions and some MilliQ water for approximately 10minutes. When that was finished degassing, I filled the reference cell with the MilliQwater, the sample cell with protein solution and the syringe with lipid solution.4

Figure 1: Control ManualFigure 1 shows the ITC (Isothermal Titration Calorimeter) Control Manual. Thisis how I changed the number of injections, the temperature of the solutions, the syringeand cell concentration, the volume, duration, spacing, and filter period. These things arevery important to check before pressing the START button. I also selected the highfeedback mode and the auto and fast equilibration options. The options in Figure 1produce 29 injections over a period of 300 seconds each (5 minutes) with a volume of10μl with a high feedback and fast/auto equilibrations. After the titration I receivedresults that looked like those shown in Figure 2 which could be processed using theOrigin 7.0 software to produce the binding isotherm as shown in Figure 3.5

Figure 2: Raw ITC data for titration of 0.025mM BSA and 0.5mM lauric acid. Thisshows the amount of heat needed to bind the lipid to the protein for this titration.6

Figure 3: Processed integrated heats for titration of 0.025 mM BSA and 0.5 mM lauricacid. Gives a visual of how many lipid molecules bind to each protein molecule. Theinflection point half-way between the horizontal plateaus at the left and right of thefigure, lies at a molar ratio of approximately 1.5.Remaking the BSA solution:The desired concentration for the BSA solution was 0.025 mM, the desiredvolume was 5ml and the molecular weight is 66000 g/mol. I was suppose to weigh 8.3mg of BSA but I actually weighed 9.6mg and dissolved it into 5 ml of 1%DMSO in PBS.So the actual concentration was 0.029 mM. This solution went into the sample cell.7

Remaking the Lauric Acid solution:The desired concentration for the lauric acid solution was 50mM , the desiredvolume was 5ml and the molecular weight in 200.32g/mol. I was suppose to weigh 50mgof lauric acid but I actually weighed 53.5mg and dissolved it into 5.25ml of DMSO anddiluted by taking 100μl of the lauric acid solution and 9900μl of PBS to make the 50mMsolution which was placed in the syringe.After making both my protein and lipid solution, I went through the exact same process.Figure 4: ITC Controls for titration of 0.5mM lauric acid into 0.029mM BSA8

RESULTS WITH NEW CONCENTRATIONSFigure 5: Raw ITC Data for titration of 0.5mM lauric acid into 0.029mM BSA:Figure 6: Integrated Heats for titration of 0.5mM lauric acid into 0.029mM BSA:9

Figure 7:ITC control manual for the BSA and myristic acid titrationFigure 7 gives the concentration of the solutions being titrated which is 0.25 for lauricacid and 0.0125 for BSA. Also, it shows where I have changed the total number ofinjection from 29 to 14 and the volume from 10 μl to 20 μl.11

Figure 8: Raw ITC for titration of 0.25 mM myristic acid and 0.0125 mM BSAFigure 9: Integrated Heats for titration of 0.25mM myristic acid and 0.0125mM BSA12

Figure 8 shows the amount of heat measured when BSA and myristic acid werebeing titrated. Even though my peaks aren’t very tall, I still seem to have gotten anisotherm. The reason I say this is because at the mid-way point of the isotherm, thebinding site of the lipid is at 1.5. The binding site is the point were the lipid binds to theprotein during the titration. This is also true for the BSA and lauric acid titration. Thepoints are scattered because the heat given off was near the instruments detection limitduring this particular titration. Figure 9 shows the processed integrated heats for thelauric acid and BSA titration. This also reflects the 1.5 binding site as shown before.Conclusion:The biding site seems to be constant at 1.5. Since I constantly had tochange the concentrations for BSA and myristic acid’s titration, I’m still working onBSA and palmitatic acid’s stoichiometry. +References:1. Inje N. Bojesen, Eigil Bojesen; Water-phase palmitate concentrations inequilibrium with albumin-bound palmitate in a biological system;Department of Biochemistry B, University of Copenhagen, PanumInstitute, 3, Blegdamsvej, DK-2200, Copenhagen N, Denmark. (Journal ofLipid Research-volume 33, 1992 p.1327)2. Alfred E. A. Thumser, David C. Wilton; The binding of natural andfluorescent lysophospholipids to wild-type and mutant rat liver fatty acidbindingprotein and albumin; Department of Biochemistry, University ofSouthampton S016 7px, UK; (Biochemistry J. 1995 p. 305)3. Anders Overgaard Pedersen, Bent Honore and Rolf Brodersen;Thermodynamic parameters for binding of fatty acid to human serumalbumin; Institute of Medical Biochemistry, Aarhus University, Aarhus,Denmark; (Eur. J. Biochem. 1990 p.190,497-502)13

4. Inje N. Bojesen, Eigil Bojesen; Binding of arachidonate and oleate tobovine serum albumin; Department of Medical Biochemistry andGenetics, Laboratory of Medical Biochemistry B, University ofCopenhagen, The panum Institute, Blegdmsvej 3, DK-2200 CopenhagenN. Denmark. (Journal of Lipid Research-volume 35, 1994)5. 3.1 Designing ITC Experiments p.214