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Chapter 18: Q29P (page 459)

18-29. Biotin-streptavidin fluorescence titration. Biotin is a cofactor in enzymatic carboxylation reactions. Biotin activatesCO2for biosynthetic reactions.


Streptavidin is a protein isolated from the bacterium Streptomyces avidinii that binds biotin with a formation constant of ~1014M-1. The biotin-streptavidin complex is widely used in biotechnology because the noncovalent complex is stable in the presence of detergents, protein denaturants, and organic solvents, and at extremes of pH and temperature.

The stoichiometry of the biotin-streptavidin complex was measured by a fluorescence titration. Fluorescein (page 453 ) covalently attached to biotin via the biotin carboxyl group fluoresces at 520 nm when irradiated at 493 nm. When biotin-fluorescein (BF) binds to streptavidin (SA), fluorescence decreases. The table gives emission intensity for addition of BF to SA and also for addition of SA to BF. Data are already corrected for dilution.

(a) Make a graph of fluorescence versus mole ratio for each titration and state the stoichiometry of binding of biotin to streptavidin.

(b) Explain the shape of each titration curve.

Short Answer

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Step by step solution

01

Define Streptavidin

a) Streptavidin is a tetramer and it binds four molecules of biotin.

b) The graph shows addition of BF to SA. In the beginning, the fluorescence increasing slowly. When SA is saturated, fluorescence increases sharply due to excess BF.

The graph shows addition of SA to BF. The fluorescence decreases until all BF is bound to SA. After that, the fluorescence is constant.

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Most popular questions from this chapter

Nitrite ionNO-2, is a preservative for bacon and other foods, but it is potentially carcinogenic. A spectrophotometric determination ofNO-2makes use of the following reactions

Here is an abbreviated procedure for the

determination:

1. To 50.0ml of unknown solution containing nitrite is added 1.00mL of sulfanilic acid solution.

2. After 10min , 2.00mL of 1 –a minonaphthalene solution and 1.00 mL of buffer are added.

3. After 15 min, the absorbance is read at 520 nm in a 5.00-cm cell.

The following solutions were analyzed:

A. 50.0 mL of food extract known to contain no nitrite (that is, a negligible amount); final absorbance =0.153.

B. 50.0mL of food extract suspected of containing nitrite; final absorbance \(=0.622\).

C. Same as B, but with 10.0μLof7.50×10−3MNaNO2added to the 50.0-mL sample; final absorbance =0.967.

(a) Calculate the molar absorptivity, of the colored product. Remember that a \(5.00\)-cm cell was used.

(b) How many micrograms ofNO-2were present in 50.0mL of food extract?

What is the wavelength, wavenumber, and name of radiation with an energy of 100 kJ/mol?

In formaldehyde, the transition n→π*(T1)occurs at 397 nm,and the n→π*(S1)transition comes at 355 nm. What is the difference in energy (kJ/mol) between the S1andT1states? This difference is due to the different electron spins in the two states.

Preparing standards for a calibration curve.

(a) How much ferrous ethylene diammonium sulfate(FeH3NCH2CH2NH3SO42⋅4H2O,FM382.15)should be dissolved in a 500mL volumetric flask with1MH2SO4to obtain a stock solution with~500μgFe/mL2?

(b) When making stock solution (a), you actually weighed out 1.627 g of reagent. What is the Fe concentration in role="math" localid="1668357579931" 500μgFe/mL2?

(c) How would you prepare 500 mL of standard containing 1,2,3,4, and5μgFe/mL2in0.1MH2SO4infrom stock solution (b) using any Class A pipets from Table 2-4 with only 500 -mL volumetric flasks?

(d) To reduce the generation of chemical waste, describe how you could prepare 50 mL of standard containing, andinfrom stock solution (b) by serial dilution using any Class A pipets from Table 2-3 with only 50mL volumetric flasks?

18-7: How do transmittance, absorbance, and molar absorptivity differ? Which one is proportional to concentration?
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