TITLE: A HABA dye based colorimetric assay to detect unoccupied biotin binding sites in a fusion protein containing avidin
AUTHORS: Sonia Mukherjee, Pierre Leblanc, Mark Poznansky, Ann Sluder

[DESCRIPTION]
HABA (4'-hydroxyazobenzene-2-carboxylic acid) dye is an anionic dye which is used to assess the biotin binding sites in avidin. Herein we describe an assay protocol to utilize the avidin binding property of HABA to assess the number of available biotin binding sites in an avidin containing HSP70 fusion protein. This approach reduces the technical and instrumentation requirements as compared to fluorescence-based assays to evaluate biotin binding. We have also miniaturized the assay using a Nanodrop detector for the readout, thereby sparing reagents.

[BEFORE_START]
Wipe the work station with 70% Ethanol. Wear gloves before handling the proteins and dyes.

[STEPS]
SECTION: Impact of biotin concentration on the displacement of HABA from Avidin in a colorimetric assay
1. Prepare Avidin-HABA complex:
Add 100 µL of a 12 micromolar (µM) Avidin stock solution to a 1.7 mL eppendorf tube containing 10.4 µL DPBS.

SECTION: Impact of biotin concentration on the displacement of HABA from Avidin in a colorimetric assay
2. 
Add 9.6 µL of a 1 millimolar (mM) HABA stock solution to obtain a final concentration of 80 micromolar (µM) HABA dye.

SECTION: Impact of biotin concentration on the displacement of HABA from Avidin in a colorimetric assay
3. Prepare a control avidin tube lacking HABA by adding 10 micromolar (µM) avidin in a final volume of 120 µL DPBS.

SECTION: Impact of biotin concentration on the displacement of HABA from Avidin in a colorimetric assay
4. Prepare a control HABA tube by adding 9.6 µL of 1 millimolar (mM) HABA to 110.4 µL of DPBS only.

SECTION: Impact of biotin concentration on the displacement of HABA from Avidin in a colorimetric assay
5. Measure the absorbance at a wavelength of 500 nm of the HABA-Avidin complex and the HABA only tube using a UV-Vis Nanodrop spectrophotometer (Thermo Scientific™ NanoDrop 2000) by pipetting 2 µLsolution onto the nanodrop pedestal.
SECTION: Impact of biotin concentration on the displacement of HABA from Avidin in a colorimetric assay
6. Prior to sample measurement, determine the spectrophotometer baseline reading using a blank solution of 2 µLDPBS.

SECTION: Impact of biotin concentration on the displacement of HABA from Avidin in a colorimetric assay
7. Add an aliquot of 10 µL of HABA-Avidin complex to each of five 0.5 mL PCR tubes.

SECTION: Impact of biotin concentration on the displacement of HABA from Avidin in a colorimetric assay
8. Add DPBS to the five tubes adequately so that after addition of D-biotin the final volume would be 15 µL.

SECTION: Impact of biotin concentration on the displacement of HABA from Avidin in a colorimetric assay
9. Add D-biotin to generate a range of D-biotin concentrations as indicated in Table 1.


Table 1: Volumes of D-biotin and DPBS added to HABA-Avidin from stock solutions to titrate out HABA in the table below. The formula C1V1=C2V2 was used to determine the volumes of D-biotin (V1) required from the stock solution (concentration C1) to achieve the final concentration (C2) in a total volume of 15 µL solution (V2).

 
Measure the absorbance at a wavelength of 500 nm of the HABA-MAV complex and the HABA only tube using a UV-Vis Nanodrop spectrophotometer.
SECTION: Displacement of HABA from biotin binding pockets of a MtbHSP70-avidin fusion protein using different concentrations of free biotin, measured using nanodrop based detection
10. Prepare MtbHsp70-avidin-HABA complex:
 Add 54 µL of 1.85 mg/mL a stock solution of a MtbHsp70-avidin (MAV) fusion protein to a 1.5 mL eppendorf tube containing 56.4 µL DPBS.

SECTION: Displacement of HABA from biotin binding pockets of a MtbHSP70-avidin fusion protein using different concentrations of free biotin, measured using nanodrop based detection
11. Add 9.6 µL of a 1 millimolar (mM) HABA stock solution for a final concentration of 80 micromolar (µM) HABA dye and 10 micromolar (µM) concentration MAV in a final volume of 120 µL.

SECTION: Displacement of HABA from biotin binding pockets of a MtbHSP70-avidin fusion protein using different concentrations of free biotin, measured using nanodrop based detection
12. Add 9.6 µL of 1 millimolar (mM) HABA to 110.4 µL of DPBS only as a control.

SECTION: Displacement of HABA from biotin binding pockets of a MtbHSP70-avidin fusion protein using different concentrations of free biotin, measured using nanodrop based detection
13. Measure the absorbance at a wavelength of 500 nm of the HABA-MAV complex and the HABA only tube using a UV-Vis Nanodrop spectrophotometer.
SECTION: Displacement of HABA from biotin binding pockets of a MtbHSP70-avidin fusion protein using different concentrations of free biotin, measured using nanodrop based detection
14. Add an aliquot of 10 µL of HABA-MAV complex each of to six 0.5 mL PCR tubes.

SECTION: Displacement of HABA from biotin binding pockets of a MtbHSP70-avidin fusion protein using different concentrations of free biotin, measured using nanodrop based detection
15. Add DPBS to the six tubes adequately so that after addition of biotin the total volume would be 15 µL.

SECTION: Displacement of HABA from biotin binding pockets of a MtbHSP70-avidin fusion protein using different concentrations of free biotin, measured using nanodrop based detection
16. Add D-biotin to the tubes to generate a range of D-biotin concentrations as indicated in Table 2.

Table 2: Volumes of D-biotin and DPBS added to HABA-MAV from stock solutions to titrate out HABA in the table below. The formula C1V1=C2V2 was used to determine the volumes of D-biotin (V1) required from the stock solution (concentration C1) to achieve the final concentration (C2) in a total volume of 15 µL solution (V2).
 

Measure the absorbance at a wavelength of 500 nm of the HABA-MAV complex and the HABA only tube using a UV-Vis Nanodrop spectrophotometer.
SECTION: Competitive displacement of HABA by PEG4-biotinylated peptides measured in colorimetric assay
17. 
Predict the water solubility of the peptides using Pepcalc (https://pepcalc.com).
SECTION: Competitive displacement of HABA by PEG4-biotinylated peptides measured in colorimetric assay
18. Dissolve the peptides to a concentration of 5 mg/mL in ultrapure water with 0.5% DPBS.
 

SECTION: Competitive displacement of HABA by PEG4-biotinylated peptides measured in colorimetric assay
19. Measure the absorbance at 500 nm of the HABA-MAV complex and the HABA only tube using a UV-Vis Nanodrop spectrophotometer at 500 nm wavelength.
SECTION: Competitive displacement of HABA by PEG4-biotinylated peptides measured in colorimetric assay
20. Add an aliquot of 10 µL of HABA-MAV complex each of to six 0.5 mL PCR tubes.

SECTION: Competitive displacement of HABA by PEG4-biotinylated peptides measured in colorimetric assay
21. Add DPBS to the six tubes for each peptide appropriately so that after addition of peptide the total volume would be 15 µL.

SECTION: Competitive displacement of HABA by PEG4-biotinylated peptides measured in colorimetric assay
22. Add biotinylated peptide to the tubes to generate a range of biotinylated peptide concentrations as indicated in Table 3.

Table 3: Volumes of biotinylated peptide and DPBS added to HABA-MAV from stock solutions to titrate out HABA in the table below. The formula C1V1=C2V2 was used to determine the volumes of biotinylated peptide (V1) required from the stock solution (concentration C1) to achieve the final concentration (C2) in a total volume of 15 µL solution (V2).
 

Measure the absorbance at 500 nm of the HABA-MAV complex and the HABA only tube using a UV-Vis Nanodrop spectrophotometer at 500 nm wavelength.
SECTION: Expected results and Data Analysis
23. The calculation for this assay is based on Beer Lambert's law (Beer's law): ΔA= εbC

ΔA is the difference in absorbance at 500 nm after addition of Biotin or biotin derivatives to the sample. 
ε is the absorptivity or extinction coefficient at the wavelength (λ).
For HABA-Avidin samples at pH 7.0, the extinction coefficient at 500 nm is equal to 34,000 M-1 cm-1. 
b is the cell path length expressed in centimeters (cm). A 10 mm-equivalent absorbance at 500 nm has a path length of 1.0 cm in Nanodrop 2000.
C is the concentration of biotin in the sample expressed in molarity (= mol/L = mmol/mL).

#Calculation 1
Absorbance at 500 nm for (HABA-avidin) reaction or (HABA-MAV) mixture= A1
Absorbance at 500 nm for (HABA-avidin) + (biotin) reaction mixture or (HABA-MAV) + (biotin) mixture= A2
ΔA=A1-A2
#Calculation 2
C (mmol/mL) = ΔA/εb

#Calculation 3
Ratio of biotin: protein
Molar concentration of bound biotin (C) / Molar concentration of original protein (MAV or Avidin)


For example, 
If 10 µM Avidin is mixed with 80 µM HABA to obtain an absorbance at 500 nm, A1= 1.298, and an unknown concentration of biotin was added to the complex and absorbance at 500 nm was measured as, A2= 0.04
ΔA= (1.298-0.04)
ΔA=1.258
C (mmol/mL) = ΔA/εb
C= 1.258/(34000*1)
C= 0.000037 mmol/ml
C= (0.000037 x 1000000) µM = 37 µM
Ratio of µmoles of biotin per µmole of Avidin= 37 µM/10 µM Avidin 
µmoles of biotin per µmole of Avidin= 3.7