Adenosine Triphosphate Atp Is A Mononucleotide

Published Date: 02 Nov 2017

Introduction

Adenosine triphosphate (ATP) 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 (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 thereby driving them 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, which comprise 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 a 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 a 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 than 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 therefore allowing 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 result 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, which in this case differential centrifugation is focus upon. 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 in all aerobic tissue and occurs within mitochondria whereby the enzyme catalyses the breakdown of succinate to fumarate forming FADHâ‚‚ in the process. Therefore 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 which are commonly used in laboratory practices. In addition centrifugation techniques, with a liver homogenate as a model will be addressed, creating fractions which are to be assayed for mitochondrial activity.

Method

Section A

Chemical Techniques

1. Quantification of ATP using Absorption by Ultraviolet Light

The quantification ATP of an unknown sample 1 (group 1) was carried out by means of ultraviolet absorption spectroscopy. The unknown sample of 100mg was diluted to make a stoke solution of 10mg/ml and for further diluted as required for subsequent absorbance spectroscopy procedures (e.g. 10x or 100x 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 is .

2. Quantification of ATP using the Ribose Content

2ml of the unknown ATP solution (group 1) was mixed with 2ml of 1% orcinol which was dissolved in 0.1% FeCl₃ in concentrated HCl. The mixture was prepared in test tube and was covered with a marble. The mixtures where then heated for 30min in a boiling water bath. The mixture was then cooled and then diluted to 4ml and the optical density was 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 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 ATP sample A with the use of a 96-well VIS microtiter plate reader. A 5 point standard curve of known BSA was created (0 -2 mg/ml) and the procedures were carried out in triplicate. It was assumed that 1mol of ribose amounted to 1mol of ATP present. Refer to Table 2 for BCA assay procedure. Refer to Table 2 of appendix for the assay procedure.

Table 1: BCA assay for the determination of protein within Bovine Serum Albumin (BSA) sample

Reagent

Volume (µl)

Sample/BSA

10

Reagent A and Reagent B* (50:1)

200

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 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 of appendix for assay procedure.

Section B

Centrifugation

Mitochondrial enzymatic activity was determined whereby 50g of 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.

*Note aliquots of each fraction (H1, S1, S2, S3, P1, P2, and P3) were retained

Fig. 1: Differential centrifugation procedure carried out on 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 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 (t₀). 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.

Results

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 the absorbance, ε is the molar extinction coefficient, c is the concentration of the sample and l is the path length (i.e. distance travelled by the light through the cuvette).

Therefore:

Therefore sample A containedof ATP

Fig 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 0.993. The unknown ATP content of sample A was determined to be 1.963 mg/ml. Note a 1:100 dilution factor was used.

Calculations:

* 1:100 dilution factor

In regards to the five point standard curve (fig.2) the ribose standard concentrations of 0.0375 and 0.05 mg/ml were 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 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 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 0.635mg/ml.

Table 2: The ATP and Protein concentration and percentage of unknown sample A

Method

Concentration

Percentage

ATP (mg/ml)

Protein (mg/ml)

ATP (%)

Protein (%)

UV Quantification by extinction coefficient

0.0219

-

0.219

-

Quantification by Ribose Content

1.9630

-

19.63

-

BCA Protein Assay

-

1.226

-

12.26

Fluorescamine Protein Assay

-

0.635

-

6.350

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):

*100

Fig 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 degrease in absorbance (fig 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 6: Bar graph representation of the succinate dehydrogenase (SDH) activity assay as seen in fig 5. Bars indicate the change in absorbance @600nm over the initial first minute of the reaction.

As observed in fig 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.

Discussion