Question

1. A 0.1% solution of lysozyme gives and absorption of 2.7 at 280 nm. (a) if the concentration is 0.01% what absorption would be measured? (b) If the absorbance of 1.0 is measured, what is the concentration?

2. Muscle contracts by sliding myosin filaments relative to actin filaments. The myosin head group hydrolyzes ATP to get the energy for its globular motor domain to move a long lever-arm domain. Suzuki et al. (1998) used GFP and its mutated relative blue fluorescent protein (BFP) in a FRET experiment to demonstrate that during the working stroke the lever-arm domain tilts against the motor domain. They constructed a fusion protein in which FP was attached to the N-terminal end of the myosin and BFP was attached to the C-terminal end. Exciting the BFP causes the GFP to fluoresce with an efficiency of 0.333 after the ATP is hydrolyzed to ADP, and the corresponds to a distance of 3.8 nm. Before the hydrolysis, the FRET efficiency is 0.082. How far do the two fluorophores move relative to each other during the working stroke?

Answer 1

1a) Use Beer’s law: A = ε*c*l where A = absorbance of the solution, ε = absorptivity co-efficient of the solution, c = concentration of the length and l = length of the solution.

Given A = 2.7 when c = 0.1%, we write,

2.7 = ε*(0.1%)*l

Let the absorbance be A’ when c = 0.01%; therefore,

A’ = ε*(0.01%)*l

Therefore,

2.7/A’ = (0.1%)/(0.01%)

===> 2.7/A’ = 10

===> A’ = 2.7/10 = 0.27

Therefore, the absorbance of the solution will be 0.27 when the concentration is 0.01% (ans).

b) Next let c’ be the concentration when A = 1.0; therefore,

1.0 = ε*c’*l

Therefore,

2.7/1.0 = (0.1%)/c’

===> 2.7 = (0.1%)/c’

===> c’ = (0.1%)/2.7 = 0.037%

The concentration of the sample is 0.037% when the absorbance is recorded as 1.0 (ans).