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Adenosine triphosphate is a mononucleotide with an adenine base and a ribose sugar to which three phosphate groups are linked. The covalent bond between the second and the third phosphate group is unstable and easily broken by hydrolysis, thereby releasing energy (Kent, 2000). ATP can therefore store energy so as to be used to drive endergonic reactions through a process of phosphorylation. Phosphorylation refers to the transfer of energy with the addition of a phosphate group from ATP to an endergonic reaction allowing for the reaction to be driven forward. ATP is therefore essential for life as it provides the much needed energy to drive a living system (Alters, 2000).
There are various ways to quantify ATP, one of which is through absorption spectroscopy by ultraviolet light. The absorbance of light, at a given wavelength, by a substance with chromophore properties results in an exponential drop in light intensity (Wilson and Walker, 2010). With the knowledge of the substances' molar extinction coefficient or the measure of how strongly the substances absorbs light at a given wavelength, the concentration of the substance can be determined with the use of the Beer-Lamberts Law:, where A refers to absorbance at the given wavelength, ε refers to the molar extinction coefficient, c the concentration of the sample and l the distance light travelled through the sample (Bansal, 2003). Purine bases such as adenine, a key component of ATP, absorb UV light between 260 and 275nm allowing for their quantification by this method.
An additional method of quantifying ATP can be carried out indirectly by an assay procedure for pentose sugar, namely ribose. As discussed earlier ATP contains one ribose molecule allowing for the assumption that 1 mol of ribose will account for 1 mol of ATP within a sample of pure ATP. Ribose in an acidic solution will result in the formation of furfural which in turn reacts with orcinol, generating a green colour. The intensity of the green colour can therefore be used to spectrophotometrically quantify ribose at 660nm and indirectly ATP, considering no additional pentose sugar contaminates are present (Nigam, 2007).
Many applications require the quantification of protein, often in a high throughput manner as can be expected within various laboratories. One such method involves the use of the bicinchoninic acid (BCA) protein assay developed by Smith et al. The assay depends on the conversion of to under alkaline conditions which in turn reacts with BCA resulting in an intense purple colour, which can be quantified through spectroscopy techniques at an absorbance of 562nm. The production of in the assay is a function of the protein concentration and the incubation time, allowing for the protein quantification (Walker, 1994). In addition, fluorimetric protein quantification can be carried out with the aid of a reactive compound; fluorescamine. Fluorescamine is a very sensitive fluorogenic reagent which reacts with primary amides to form fluorescent pyrrolinones. This results in a green-yellow fluorescence which can be quantified as a function of protein amide groups (Rost, 1995), such as the terminal protein amide groups and ε-amino group of lysine (Rosenthal and Wright, 2005).
Other general techniques often used within a biochemical laboratory are centrifugation techniques. In this case differential centrifugation is the focus. The process is based upon the differing sedimentation rates of biological particles as a function of differing size and density. For example, this allows for the division of crude homogenates into various fractions of organelles, membrane vesicles and other structural fragments by means of a stepwise increase in the applied centrifugal field and centrifugation times (Wilson and Walker, 2010). One of the classic examples is the stepwise separation of liver homogenate to isolate liver mitochondria, whereby the effectiveness of the technique can be analyzed through a succinate dehydrogenase (SDH) assay. Succinate dehydrogenase exists ubiquitously within mitochondria in all aerobic tissue whereby the enzyme catalyses the breakdown of succinate to fumarate forming FADHâ‚‚ in the process. The breakdown of succinate can be used as a mitochondrial indicator by addition of blue/purple redox dye namely dichlorophenol indophenols (DCPIP). Blue DCPIP acts as a hydrogen acceptor from FADHâ‚‚ (i.e. product of the SDH enzyme) resulting in a loss of colour during the process, which can be directly quantified spectrophotometrically at an absorbance of 600nm and thereby related to mitochondrial concentration (Nigam, 2007).
The aim of the experiment is to analyze various techniques to determine ATP and protein concentrations of unknown ATP samples A. In addition, centrifugation techniques with a chicken liver homogenate as a model will be addressed, creating fractions which are to be assayed for mitochondrial activity.
1. Quantification of ATP using Absorption by Ultraviolet Light
The quantification ATP of an unknown ATP sample A (group 1) was carried out by means of ultraviolet absorption spectroscopy. The unknown sample of 0.45g was diluted to make a stock solution of 10mg/ml and for further diluted as required for subsequent absorbance spectroscopy procedures (e.g. 1:10 or 1:100 dilution). The absorbance spectrum from 220 to 310nm of the unknown ATP sample was performed and the content of the ATP was determined from the optical density at 295nm. The molar extinction coefficient of ATP at pH 7 at 295nm was taken to be.
2. Quantification of ATP using the Ribose Content
2ml of the unknown ATP sample A solution was mixed with 2ml of 1% orcinol which was dissolved in 0.1% FeClâ‚ƒ in concentrated HCl. The mixture was prepared in test tubes and sealed with cotton bungs. The mixtures were then heated for 30min in a boiling water bath. The mixture was then cooled and then diluted to 4ml. The optical densities of the samples were then observed at 660nm. A standard curve was also created ranging from 0.01 - 0.1 mg/ml whereby the unknown sample was substituted for known concentrations of ribose. The whole procedure for determining the ribose content was carried out in triplicate. Refer to Table 3.1 for assay procedure within the appendix.
Biochemical Techniques - Protein Determination Methods
1. VIS Microtiter Plate Quantification of Protein using the Bicichonic Acid (BCA) assay
A BCA assay procedure was carried out to determine the protein content of unknown ATP sample A with the use of a 96-well VIS microtiter plate reader. A five point standard curve of known BSA was created (0 -2 mg/ml). Refer below to Table 1.1 for BCA assay procedure. Refer to Table 3.2 of appendix for the assay procedure.
Table 1.1: BCA assay for the determination of unknown ATP sample A within Bovine Serum Albumin (BSA) standard curve
Reagent A and Reagent B* (50:1)
Incubated at 37°C for 30min. Absorbance read at A540nm
*Refer to appendix for constituents of reagent A and B
2. Fluorimetric Quantitation of Protein using the Reactive Compound Fluorescamine
A five point standard curve of BSA was constructed ranging from 0 - 500µg/ml, diluted in phosphate buffered saline (PBS) at pH 7.4 to determine the unknown ATP sample A protein concentration. 150µl aliquots were then transferred to a black 96-well microtiter plate followed with the addition of 50µl of 3mg/ml fluorescamine dissolved in acetone. The plate was then shaken for 1min followed by fluorescence determination with a 355nm excitation filter and a 460nm emission filter. All samples were carried out in triplicate. Refer to Table 3.3 of appendix for assay procedure.
Mitochondrial enzymatic activity was determined whereby 50g of chicken liver tissue was obtained and minced before being diluted in 10ml of sucrose isolation medium (300mmol/l sucrose: 0.5mmol/ EDTA at pH 7.4) per gram of tissue. The sample was then homogenized with a co-axial homogenizer. The mortar and pestle was then fixed to a drill resulting in relative mortar movement to the pestle, rotating at 2400rpm for 8-10 complete strokes. Procedures were performed at 4°C. Centrifugation of the suspension was then carried out as observed in Figure 1.1. However, the last centrifugation step was altered to be 40000xg for 120min as opposed to 100000xg for 120min due to the rotor of ultracentrifuge only being rated to 40000xg.
*Note aliquots of each fraction (H1, S1, S2, S3, P1, P2, and P3) were retained
Fig. 1.1: Differential centrifugation procedure carried out on chicken liver homogenate
A succinate dehydrogenase (SDH) assay was performed on the homogenate, supernatant and pellet samples obtained during differential centrifugation of the liver sample. Assay medium was created as can be seen in the appendix (Table 3.4) to be used in the SDH assay. For each sample 2.8ml of the assay medium was pre-warmed to 37°C followed by the addition of 0.2ml of the dilute enzyme within the homogenate, supernatant or pellet samples and the absorbance was determined at 600nm. This was taken to be time zero (tâ‚€). The subsequent absorbance values were recorded at 1min time intervals for a total of 5min. A progress curve was then plotted for each fraction to determine the change in absorbance over time. The sucrose isolation media was used as a blank.
1. Quantification of ATP using Absorption by Ultraviolet Light:
Absorption of ultraviolet light by ATP is a function of the adenine group. The given molar extinction coefficient of ATP at pH 7 at 295nm is. The optical density of sample A was determined to be 0.664nm at an ABS of 295nm:
Therefore the Beer-Lamberts law states:
Where A is the absorbance, ε is the molar extinction coefficient, c is the concentration of the sample and is the path length (i.e. distance travelled by the light through the cuvette).
Therefore sample A containedof ATP, however the sample pH was higher and did not correlate to the pH of 7 that allowed for the assumption of a molar extinction coefficient of.
Fig. 2.1: The absorbance spectrum of unknown ATP sample A ranging from 210nm to 290nm. An absorbance peak of 260nm was observed that correlate to, and therefore confirms the presence of ATP.
Fig 2.2: Standard curve for the quantification of ATP using the ribose content. It was assumed that 1mol of ribose amounted to 1mol of ATP. The equation for the best fit line: with a linear regression of 0.901. The unknown ATP content of sample A was determined to be 2.425 mg/ml. Note a 1:100 dilution factor was used.
* 1:100 dilution factor
In regards to the five point standard curve (fig.2.2), the ribose standard concentrations of 0.05 mg/ml was omitted. This was due to spectrometry errors as the instruments showed varying absorbencies in the higher concentrations values which did not correspond to expected results after multiple replicates. It is believed this was due to instrument damage during renovations to the Nelson Mandela Metropolitan University (NMMU) biochemistry department.
Fig 2.3: Standard curve for the VIS microtiter plate protein quantification of the unknown sample A, using the bicinchonic acid (BCA) assay. The equation for the best fit line: with a linear regression of 0.998. The unknown protein content of sample A was therefore determined to be 1.226mg/ml. Note a 1:10 dilution factor was used.
Fig 2.4: Standard curve for the quantification of unknown protein of sample A by means of the fluorescamine assay. The equation for the best fit line: with a linear regression of 0.977. The unknown protein content of sample A was therefore determined to be 3.191mg/ml.
In regards to fig2.4 the final point on the curve that amounted to a concentration of 500µg/ml, was omitted so as to increase the correlation coefficient closer to 1.0. This was due to high standard deviations and emission values which did not correspond to the concentration.
Table 2.1: The ATP and Protein concentration and percentage of unknown sample A
UV Quantification by extinction coefficient
Quantification by Ribose Content
BCA Protein Assay
Fluorescamine Protein Assay
High discrepancies between ATP and protein quantification was observed between the given techniques, as large percentage variations were evident.
Calculations (e.g. UV Quantification by extinction coefficient):
Fig 2.5: Succinate dehydrogenase (SDH) activity assay indicating the change in absorbance @600nm as a function of mitochondrial concentration within the given fractions. The following fractions were assayed: the homogenate (H), pellet 1, 2 and 3 (P1, P2, and P3) and the supernatants 1, 2, and 3 (S1, S2, and S3).
The greatest decrease in absorbance (fig 2.5) was observed to be the homogenate fraction (H), followed by the first pellet (P1). P1 was obtained after the centrifugation step at 3000xg for 20min. The subsequent fractions did not yield high decreases in absorbance as compared to the H and P1 fractions and therefore did not show significant mitochondrial activity.
Fig 2.6: Bar graph representation of the succinate dehydrogenase (SDH) activity assay as seen in fig 2.5. Bars indicate the change in absorbance @600nm over the initial first minute of the reaction.
Determination of succinate dehydrogenase activity (e.g. Fraction H):
As observed in fig 2.5 the highest degree of activity was observed for the homogenate fraction (H) followed by the first pellet (P1). The subsequent fractions did not show high mitochondrial activity in retrospect and therefore were not significant.
The quantification of ATP of the unknown sample A by ultraviolet light as a function of Beer-Lamberts Law showed that 0.219% of the sample comprised of ATP. This was assumed that the conditions for absorption of ATP was constant and related to previous studies where the molar extinction coefficient was determined at pH 7. The unknown sample A did not however, have a pH of 7 but one that was considerably higher which would lead to possible inaccuracies in the quantification. The altering of the samples environment due to pH inconsistencies results in protonation/deprotonation of the sample and therefore affects the distribution of electrons within the chromophore. This will correlate to differences between absorbance spectrums at varying pH values (Sheehan, 2009). However, when taking the observed absorbance spectrum of the unknown sample A into account (fig.2.1), there were no significant shifts in the spectrum. This would indicate no true alterations the structure of the sample occurred due to the incorrect pH value and ill effect on the observed ATP concentration calculated. The method for pH quantification assumed that the molar extinction coefficient for ATP was at a wavelength of 295nm, however this is incorrect. A molar extinction coefficient of this value for ATP correlates to a wavelength of 259nm and not one of 295nm therefore quantification of the sample was measured incorrectly and cannot be considered accurate (Gerstein, 2004). The absorption spectrum (fig.2.1.) for ATP strengthens this conclusion as an absorbance peak was observed at 260nm (i.e. very close to 259nm) and not near 295nm. Due to these inaccuracies the results obtained for the unknown ATP concentration cannot be consider valid and therefore determination relies on the ATP quantification by a function of ribose content and the orcinol assay.
The orcinol assay for ribose content yielded results that indicated that 24.25% of the unknown sample A comprised of ATP. However, these results in addition cannot be considered accurate. The spectrophotometery instrumentation used produced varying results primarily at the higher absorbance values at a wavelength of 660nm. It was determined that this was due to decalibration and/or damage to the instrumentation during renovations to the laboratory where the instruments were stored. This resulted in the removal of the higher concentration points of the standard curve to obtain a respectable correlation coefficient (fig.2.2.). For an accurate result in this case the concentration of the unknown sample is required to fall between the first three concentration/absorbance values of the standard curve. However, the true amount of damage sustained by the spectrophotometery instrumentation cannot be fully assessed and therefore the results obtained should not be considered.
The quantification of ATP of unknown sample A by both methods namely; quantification of ATP by absorption of UV light and the quantification of ATP by the determination of the ribose content were not accurate. Therefore, modification of the methods should be carried out whereby the pH is more stringently controlled and quantification should be carried out at the correct wavelength (i.e. 259nm) in the case of the first method. In the case of the second method, the accuracy of the instruments used should be verified prior to use. There are however, other methods to determine the ATP content of a sample more accurately, one of which is high performance liquid chromatography (HPLC). Manfredi et al. in their measurements of ATP in mammalian cells optimized a HPLC protocol for the quantification of ATP, ADP and AMP. With the use of ion-pair reverse-phase HPLC system with a phosphate-buffered acetonitrile gradient mobile phase the ATP of the unknown sample A can be quantified (Manfedi et al., 2002). This method will be more effective as contaminates would not interact and interfere with the ATP due to varying dissociation coefficients allowing for only ATP quantification.
Protein determination by the bicinchonic acid assay has been determined to be sensitive to 0.5-10µg protein/ml when performed as a microassay, and is in most cases chosen over the Lowry assay method as it is more tolerable to interfering compounds (Walker, 1994). Generally the absorbance used for the BCA assay is 562nm however, in this case, an absorbance of 540nm was used. In regards to filter based plate readers a wavelength range of 540 to 590nm can be used when performing the reaction without a large degree of sensitivity loss (Burgess and Deutscher, 2009). Therefore, any inaccuracies that may have occurred due to the chosen wavelength do not have to be considered in this case. From this method it was determined that 12.26% of the unknown ATP sample A contained protein, however an absolute concentration of protein cannot be determined. This is due to the fact that the BCA assay is dependent on the amino acid composition of the protein, and therefore discrepancies will arise when using a bovine serum albumin standard curve (BSA). That is to say the BSA amino acid/peptide composition will differ slightly to that of the unknown protein sample (Walker, 1994).
Fluorescamine assay for protein determination in comparison to the BCA assay procedure is considerably more sensitive. Fluorescamine allows for the quantification of proteins at levels as low as 25-50pmoles (Lawrence and Frei, 2000). It was determined from the fluorescamine assay that 31.91% of the unknown sample A comprised of protein which is over twice that determined by the BCA assay. The amount of protein present was determined through the 1:100 dilution of the unknown ATP sample A as fluorescamine has a greater sensitivity at lower concentrations and this dilution yielded results with the lowest standard deviation. The fluorescamine assay was carried out at a pH of 7.4 which is not ideal for maximum sensitivity as the low pH can strongly influence the fluorescamine intensity. Maximum sensitivity occurs at a pH of 9 (Keyes, 2000). For best results the method should be altered to account for the change in pH and compared to the previous fluorescamine assay results on the unknown sample A. This will allow for a comparison that will show whether or not the observed unknown sample A protein concentration was indeed altered by the low pH. At this stage however, it can be assumed that the protein content of the unknown sample A was in the region of about ±20% when considering both BCA and the fluorescamine assay results.
In conclusion, the determination of the ATP content of the unknown sample A by the two stipulated methods was unsuccessful. This was due to incorrect absorbance values in the case of ultraviolet quantification and faulty spectrophotometery instruments. In the case of the protein determination of unknown sample A by the two stipulated methods discrepancies between the methods arose which didn't allow for absolute protein quantification. However, a rough estimate could be determined but would be inadequate for most subsequent applications with the sample.
Differential centrifugation of liver homogenates for mitochondrial isolation generally follows a very similar procedure, where the homogenate is first centrifuged at a low centrifugal force to remove cell debris and nuclei (700xg). The centrifugation of the subsequent supernatant is carried out at a much higher centrifugal force (e.g. 20000g) to separate most of the mitochondria from the fraction. This would result in the second pellet having the highest mitochondrial activity in comparison to any possible fractionation steps carried out (Ghosal and Srivastava, 2009). In regards to the method used in this experiment the first centrifugation step was performed at a high centrifugal force resulting in most of the mitochondria present in the first pellet as opposed to the second pellet in the general isolation techniques. This would account for the indicator enzyme, succinate dehydrogenases' high observed activity in pellet 1. The homogenate also showed very high mitochondrial activity as to be expected as it would contain the highest concentration of mitochondria. However, in relation to pellet 1 the homogenate should not have as high a level of mitochondrial purity due to the presence of the cell debris and nuclei which would interact and interfere with enzymatic reactions. The low levels of succinate dehydrogenase activity in the subsequent fractions after pellet 1 would be an indication of mitochondrial cross contamination during the stepwise fractionation procedure. That is to say residual mitochondria remained within the supernatant of pellet 1. This would be due to the relatively low centrifugal force applied to the homogenate to create the mitochondrial pellet allowing for residual mitochondria to remain in the supernatant.
For a higher isolation yield of mitochondrial isopycnic centrifugation should have been carried out allowing for a purer mitochondrial product. The fraction enriched in mitochondria (i.e. pellet 1) would be subjected to resuspension in a 20% saccharose solution and then placed in a centrifugation tube which contains a saccharose gradient. The gradient would be required to be 20% saccharose at the top of the centrifugation tube and 50% saccharose at the bottom. High speed centrifugation will therefore separate the mitochondria from the lysosomes and peroxisomes as a function of density yielding higher resolution (Vignais and Vignais, 2010).
In conclusion it was observed that mitochondrial isolation from a chicken liver sample occurs in the first or second fraction of stepwise differential centrifugation, depending on protocol variations. This method of isolation is sufficient considering a low level of purity is acceptable for the desired application.
Table 3.1: Assay procedure for determining the ribose content of unknown ATP sample 1
Ribose std conc.
Cover tubes, heat for 30min in boiling water bath, cool, dilute to 4ml
* Ribose Std. = 0.05mg/m
Table 3.2: The procedure for determination of the protein content of unknown sample A with use of bicinchonic acid assay
BSA std* (µl)
BSA (Unknown) (µl)
Reagent A and B (µl)
Incubate for 30min at 37°C
* BSA Std. = 2mg/ml
Table 3.3: Assay procedure for determination of the protein content of bovine serum albumin (BSA) with use of reactive compound fluorescamine
BSA Conc. (µg/ml)
BSA std* (µl)
BSA (Unknown) (µl)
Plate shaken for 1min and read at excitation of 355nm and an emission of 460nm
* BSA Std. = 500µg/ml
Table 3.4: Constituents of the assay medium required for the succinate dehydrogenase (SHD) assay
Phosphate buffer, pH 7.4
Table 3.5: Succinate dehydrogenase activity assay on the given liver fractions
Change in Abs/min
*Note that the concentrated fraction samples were used for all activity assays and that absorbance was measured at 600nm.
Diluting unknown sample A:
Orcinol 1% w/v, FeClâ‚‚ 0.1% w/v (600ml):
Dilution of 100mg/ml ribose stock solution (300ml):
Therefore 0 .15ml stock solution to 299.85ml of dHâ‚‚O
Reagent A (40ml):
1.00%: 0.4g BCA salt
2.00%: 0.8g Sodium carbonate
0.10%: 0.04g Sodium tartrate
0.40%: 0.16g Sodium hydroxide
0.95%: 0.38g Sodium bicarbonate
E.g. (1.00% BCA salt)
Reagent B (10ml):
4.00% copper sulphate
Sucrose isolation media (4L):
Assay Medium (600ml):
10mM Succinate - 708.5mg
2.0mg/ml DCPIP - 1200mg
10mM Phosphate Buffer, pH 7.4 - 1.014g (650ml made)
2mM KCN - 78.1mg
10mM CaClâ‚‚ - 882.1mg
0.5mg/ml Albumin - 300mg
E.g (10mM Succinate)
PBS - 10mmol PO4 and 0.9% NaCl:
Distilled H2O: up to 40ml