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Comparison of Protein Concentration in Leaves Between Sun and Shade Plants

Essay by   •  March 19, 2016  •  Lab Report  •  1,047 Words (5 Pages)  •  2,013 Views

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COMPARISON OF PROTEIN CONCENTRATION IN LEAVES BETWEEN SUN AND SHADE PLANTS.

Introduction:

RuBP carboxylase (Rubisco) is the major enzyme catalysing the reaction of fixing CO2 in the atmosphere to RuBP during photosynthesis. When the rate of photosynthesis increases due to high light level, it is predicted that more Rubisco is available in leaf. As a result, protein concentration in leaf rises in Sun leaves as 80% of leaf protein is Rubisco.

One of the methods to determine protein concentration is using Biuret test. Biuret reagent contains a Copper (II) ion in alkaline solution. In presence of protein with more than 2 peptide bonds (-NH-CO-), a violet complex is formed. The intensity of the colour can easily be determined by spectrophotometer. Biuret test provides a tool to take measurement of protein concentration indirectly through the reading of absorbance to which protein amount is proportional.

This experiment is designed to demonstrate the relationship between protein concentration and absorbance via standard cure which is then used to work out the protein amount in silverbeet leaf extracts. Further comparison between samples from Sun and Shade conditions is also performed.

Hypothesis: Protein concentration in Sun leaves is higher than Shade ones.  

Method:

 A standard protein solution which is bovine serum albumen (BSA) with concentration of 10mgml-1; a phosphate buffer; biuret reagent and 2 silverbeet leave suspensions (Sun and Shade) were given. BSA solution (1-6) was prepared according to Table 1 into 6 tubes, each with a different protein concentration. The same step was taken with leave suspensions as shown in Table 2. 4ml of Biuret reagent was added to each tube. Cap all 10 tubes and mix the contents well. The tubes were left at room temperature for 25 minutes before the absorbance measurement was taken by spectrophotometer.

The final concentrations of solutions in tube 1-6 (C2) were calculated with below general equation:

 Concentration pre-dilution  x  Volume pre-dilution   =  Concentration post-dilution  x  Volume post-dilution

Or                                 C1V1 = C2V2       in which C1 = 10mg ml-1; V2 = 1.0ml

Results:

Table 1 shows that as protein concentration increases, absorbance increases. The relationship is further demonstrated by Figure 1 and a line of best fit has been drawn. 

Tube

1

2

3

4

5

6

Buffer (ml)

1.0

0.8

0.6

0.4

0.2

0.0

BSA Protein solution (V1)

(10mg ml-1)         (ml)

0.0

0.2

0.4

0.6

0.8

1.0

Biuret reagent (ml)

4.0

4.0

4.0

4.0

4.0

4.0

Total volume (ml)

5.0

5.0

5.0

5.0

5.0

5.0

Final concentration (mg ml-1) (C2)

0.0

2.0

4.0

6.0

8.0

10.

Absorbance

0.000

0.129

0.246

0.345

0.506

0.621

Table 1: Experimental protocol for protein standard solutions for constructing standard cure.

.[pic 1][pic 2][pic 3][pic 4]

Original procedure

Modified procedure

Sun

Shade

Sun

Shade

Protein solution (ml)

1.0

1.0

0.2

0.2

Buffer (ml)

_

_

0.8

0.8

Biuret reagent (ml)

4.0

4.0

4.0

4.0

Absorbance

1.684

1.451

0.344

0.320

Protein concentration derived from standard cure (mg ml-1)

_

_

5.59

5.46

Protein concentration of original, unmodified solution (mg ml-1)

_

_

27.95

27.30

 Table 2 indicates that the protein concentration in Sun leave is 2.3% higher than Shade one. The Shade/Sun ratio is 0.98.

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