Isolation of Cytochrome C from Beef Heart BCHM 3100K-02

Isolation of Cytochrome C from Beef Heart BCHM 3100K-02 John T. Johnson April 15, 2013 Dates Performed: Partner: Instructor: 01-Mar-2013 08-Mar-2013 22-Mar-2013 29-Mar-2013 05-Apr-2013 Anthony Ferrante Dr. Candace Timpte 1 Introduction 1.1 Cytochrome C (a) Cytochrome c with heme prosthetic group shown in grey, and a central iron atom shown in orange [2]. (b) The electron transport chain with cytochrome c shown between Complex III and Complex IV [1]. Figure 1.1: Illustrations of cytochrome c Building on the work of MacMunn, Keilin was the first to coin the name cytochrome for a cellular pigment found not only in parasitic insects and worms, but also in vertebrates, yeast, and some higher plants. Its ubiquity led Keilin to declare that cytochrome is, one of the most widely distributed respiratory pigments [6]. 1

Cytochrome c (c is for complex) is an important component of the electron transport chain (ETC) as illustrated in Figure 1.1b on the preceding page. Cytochrome c is found in all living organisms and is evidence of common evolutionary decent. Cytochrome c contains a bound iron molecule (See Figure 1.1a on the previous page) which enables it to transport one electron from ETC Complex III to ETC Complex IV in the mitochondrial membrane [10]. In this series of experiments a variety of standard lab techniques were used to isolate, purify and quantify cytochrome c using beef heart as the source. 1.2 Homogenization Though use of the Waring blender is known to cause denaturation of enzymes and oxidation of compounds through the introduction of air into the sample, and caution has been urged in the use of the blender to prepare samples [8], there was little cause to suspect that the cytochrome c extracted from beef heart would be damaged by use of the blender. 1.3 Gel Filtration Chromatography GFC separates molecules based on size [10]. The interaction of molecules with the Sephadex beads in the column effectively sort the molecules based on size. Larger molecules interact less and leave the column before smaller molecules[4]. 1.4 Ion Exchange Chromatography IEC separates molecules based on their charge. Generally, a column with a matrix of opposite charge to the molecule of interest is used. The molecules of interest are attracted to the matrix due to the opposing charge. An eluent with a different ph is used to effect the release of the molecule of interest from the matrix. The resulting eluant is collected in fractions [10]. In this experiment, Amberlite CG-50 was used as a cation resin. Its negative charge attracts the positively charged cytochrome c, just as the phospholipids of the mitochondrial membrane do. Washing with a high-salt buffer effects the release of the cytochrome c from the resin since the salt ions are attracted to the Amberlite, thus displacing the cytochrome [7]. 1.5 Bradford Protein Assay In the Bradford assay, the maximum absorption wavelength of Coomassie blue is shifted from 465 nm to 595 nm due to the binding of protein to the Coomassie blue. This spectral shift is proportional to the amount of protein that binds, hence, the amount of protein in a sample can be assayed by the spectral shift of Coomassie blue [10]. 2

2 Procedure Please see Isolation of Cytochrome C from Beef Heart for step-by-step instructions. Modifications are listed below. Mechanical lysis and extraction 1 ml of beef heart homogenate was not retained. Mass of tissue in homogenate was discarded after incubating on ice. Redox assay Redox assay was performed for pure cytochrome c only. 3

3 Results 3.1 Homogenization Volume of homogenate was 100 ml plus a mass of tissue that was discarded. Total volume of supernatant: 18 ml. 3.2 Ion Exchange Chromatograhpy Ion exchange filtration yielded the fractions shown in Table 3.1, and which are graphed in Figure 3.1. Fractions one and two were pooled. Table 3.1: Fractions collected from ion exchange filtration Fraction A 550 Volume Pooled? 1 1.204 0.55 ml pooled 2 0.393 0.80 ml pooled 3 0.066 1.00 ml 4 0.024 0.80 ml 5 0.020 1.00 ml 6 0.010 0.45 ml 1.500 Sample Absorbance 1.125 A_550 0.750 0.375 0 0 1 2 3 4 5 6 Tube Figure 3.1: Absorbances for ion exchange fractions. Fractions 1 and 2 were pooled due to their high absorbance which indicates a relatively high concentration of protein. 4

3.3 Gel Filtration Chromatograhpy Fractions collected from gel filtration chromatography are shown in Table 3.2 and are graphed in Figure 3.2. Fractions two and three were pooled. Table 3.2: Fractions collected from gel filtration Fraction A 550 Volume Pooled? 1 0.437 0.30 ml 2 0.785 0.30 ml pooled 3 0.479 0.30 ml pooled 4 0.385 0.45 ml 5 0.262 0.45 ml 6 0.174 0.45 ml 7 0.126 0.50 ml 8 0.093 0.40 ml 9 0.069 0.60 ml 10 0.030 0.65 ml 11 0.009 0.55 ml 12 0.008 0.50 ml 13 0.009 0.70 ml 0.8 Sample Absorbances 0.6 A_550 0.4 0.2 0 0 3 6 9 12 15 Tube Figure 3.2: Absorbances for gel filtration chromatography fractions. Fractions 2 and 3 were pooled due to their high absorbance which indicates a relatively high concentration of protein. 5

3.4 Bradford Protein Assay Absorbances of protein standards are shown in Table 3.3 and are graphed in Figure 3.3. Table 3.3: Bradford assay of standards Concentration (mg/ml) A 550 0.125 0.005 0.250 0.059 0.500 0.147 0.750 0.208 1.000 0.294 1.500 0.364 2.000 0.490 0.60 Concentration vs. Absorbance y = 0.2494x + 0.0056 R² = 0.9797 0.45 A_550 0.30 0.15 0 0 0.5 1.0 1.5 2.0 mg/ml Figure 3.3: Protein Standard Curve. Concentrations of standards make up the x axis, while their absorbances make up the y axis. The resultant equation for the best-fit linear line is shown. By setting y equal to the absorbance of an unknown and solving for x, the protein concentration for the unknown can be ascertained. The R 2 (coefficient of determination) value of 0.98 means that 98% of the absorbance value for each sample can be explained by the sample s protein concentration. The remaining 2% is due to unknown variables [9]. 6

The absorbances of samples of pure cytochrome c, beef heart ion exchange and beef heart extract are shown in Table 3.4. Table 3.4: Bradford assay of samples (For calculations, see Section 4.1 on page 9) Sample Dilution A 550 Concentration Pure 1:1 0.484 1.92 mg/ml Pure 1:10 0.037 1.26 mg/ml BHIE 1:10 0.050 1.78 mg/ml BHIE 1:100-0.033 invalid BHE 1:10 0.408 16.13 mg/ml BHE 1:100 0.013 2.97 mg/ml BHE 1:1000-0.031 invalid 3.5 SDS-PAGE The results of running an SDS-PAGE gel of beef heart extract, flow through, ion exchange, and pure cytochrome c are shown in Figure 3.4a. (a) SDS-PAGE original gel. Lanes from left to right are: beef heart extract, flow through, ladder, ion exchange, pure cytochrome c (PCC). Note absence of expected band in the PCC lane. See Error Analysis Section 5.1 on page 11. (b) SDS-PAGE used in lieu of distorted original gel. The left lane is pure cytochrome c, next is the ion exchange product, followed by the beef heart extract. The right lane is the ladder. Figure 3.4: SDS-PAGE gels 7

1000 Distance vs. Molecular Weight Molecular Weight (kd) 100 10 y = 2508.2x-0.9305 R² = 0.9876 1 0 50 100 150 200 250 300 Distance (px) Figure 3.5: Distance vs. Molecular Weight based on the ideal gel shown in Figure 3.4b on the previous page. 8

3.6 Redox Protein Assay The absorbances for the redox assay are shown in Table 3.5. Table 3.5: Absorbances for redox assay of protein concentration for pure cytochrome c sample Oxidation State Wavelength (nm) Absorbance (%) Oxidized Reduced 550 0.153 542 0.193 550 0.350 542 0.250 Table 3.6: Cytochrome C Purification Table Fraction Vol [Protein] Total Protein [Cyt c] Total Cyt c Sp. Act. Overall Yield (ml) (mg/ml) (mg) (mg/ml) (mg) (Cyt c/prot) (%) BHE 18.0 9.55 171.90 100.00 BHIE 1.3 1.78 2.31 1.34 PCC 1.3 1.59 2.07 1.59 2.07 1.20 4 Calculations 4.1 Calculating Protein Concentration The equation for the linear curve fit to the plot of standard protein absorbances is shown in Equation 4.1. absorbance = 0.2494 concentration + 0.0056 (4.1) Solving the equation for concentration gives Equation 4.2. concentration = absorbance 0.0056 0.2494 (4.2) To calculate the concentration of Pure 1:1 (for example), Equations 4.3 and 4.4 are used. The remaining calculated values are shown in Table 3.4. absorbance 0.0056 OriginalConcentration = dilution 0.2494 (4.3) 0.484 0.0056 OriginalConcentration = 1 = 1.92 mg/ml 0.2494 (4.4) 9

4.2 Calculating Molecular Weight From Figure 3.5 on page 8 we obtain Equation 4.5. MolecularW eight = 2508.2distance 0.9305 (4.5) Substituting the prominent bands on the gel seen in Figure 3.4b on page 7 into Equation 4.5, the following molecular weights were calculated. Table 4.1: Molecular Weight Calculations Calculated Lane Distance Molecular Weight (kda) Pure Cyt-C 256 14.4 Ion Exchange Beef Heart Extract 66 50.8 239 15.4 66 50.8 239 15.4 4.3 Redox Assay of Protein Concentration The general formula for calculating protein concentration using differing absorbances in the oxidized and reduced states is given in Equation 4.6. mm = (A 550r A 542r ) + (A 542x A 550x ) δɛ 550r δɛ 550x dilution (4.6) Substituting the values from Table 3.5 gives Equation 4.7 mm = (0.350 0.250) + (0.193 0.153) 29.4 10 3 9.8 10 3 1.7 = 12.143 mm (4.7) 10

5 Conclusion The protein purification was a success until the final gel filtration chromatography or the SDS-PAGE gel. As discussed in the error analysis in Section 5.1, the absence of a band in the PCC lane at the predicted position of cytochrome c indicates the failure of one or both of these steps. Using the reference gel, it can be seen that as the sample progressed through the purification steps, the number of bands was reduced until the final PCC lane shows one prominent band. This indicates that the purification was successful. The falloff in intensity of the bands shows there was some loss of protein when proceeding through the purifications. Our protein was not purified as discussed above and in Error Analysis. Using the reference gel: The prominent band in lane 1, the Pure Cytochrome C lane, was calculated to be 14.4 kda. This is close to the value of 11.7 kda found in an extensive UniProtKB search [3]. The protein appears to be pure, as there are no other bands in the lane. 5.1 Error Analysis All Bradford absorbances were read at 550 nm, rather than the optimal 595 nm. According to tests reported in a tech report from Thermo Scientific, this error on our part would result in suboptimal, but still valid, absorbances [5]. The graph for multiple wavelengths from the report is reproduced in Figure 5.1. Figure 5.1: BSA absorbance curves at different wavelengths [5]. 11

Collection of one or more fractions prior to collecting the first fraction listed in Table 3.1 on page 4 would have yielded a better graph than the one shown in Figure 3.1 on page 4, and more certainty when pooling fractions. However, this shouldn t have contributed a significant amount of error. I m not sure what happened to our original gel shown in Figure 3.4a on page 7, perhaps something in the desiccation process caused it to distort. As a result, all calculations were performed using the reference gel shown in Figure 3.4b on page 7, which is somewhat distorted and probably led to the difference in calculated vs. published protein size as noted in Section 5 on the previous page. Also of note is the virtual absence of a band at 11.7 kda for cytochrome c in the pure cytochrome c lane of the original gel shown in Figure 3.4a on page 7. Cytochrome c appears to be present in the samples taken from the steps leading to the final gel filtration, so the error was apparently in the gel filtration or in running the SDS-PAGE gel. It is possible that we washed the gel filtration column with high-salt buffer. Yields from protein purification were unusually small at 1.34% for BHIE and 1.20% for PCC. This might be explained by washing the column with high-salt buffer, rather than low-salt buffer. This would result in washing away most of the cytochrome c before fraction collection began. References [1] Sep 2007. http://en.wikipedia.org/wiki/file:mitochondrial_electron_ transport_chain---etc4.svg. [2] Sep 2011. http://en.wikipedia.org/wiki/file:cytochrome_c.png. [3] Apr 2013. http://www.uniprot.org/uniprot/p62894. [4] P. Andrews. Estimation of the molecular weigths of proteins by sephadex gel-filtration. Biochemistry Journal, 91:223 233, August 1963. [5] Authorized Associate. Determine acceptable wavelengths for measuring protein assays. Technical report, Thermo Scientific, 2010. [6] D. Keilin. On cytochrome, a respiratory pigment, common to animals, yeast, and higher plants. Proceedings of the Royal Society of London, pages 312 339, 1925. [7] Boris F Krasnikov, Nickolay S Melik-Nubarov, Lubava D Zorova, Alevtina E Kuzminova, Nickolay K Isaev, Arthur J L Cooper, and Dmitry B Zorov. Synthetic and natural polyanions induce cytochrome c release from mitochondria in vitro and in situ. Am J Physiol Cell Physiol, 300(5):C1193 203, May 2011. [8] R Stern and LH Bird. The use of the waring blender in biochemical work. Biochemical Journal, 44(5):635 637, July 1948. [9] A. Marty Thomas. R squared and linear regression best-fit line. email, Apr 2013. [10] D Voet, JG Voet, and CW Pratt. Fundamentals of Biochemistry. John Wiley and Sons, third edition, 2008. 12