Degree of labeling (DOL)

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1 Degree of labeling (DOL) A written explanation for the determination of the degree of labeling when using NuLink reagents. The DOL is the average number of reagent molecules that have been covalently attached to the sample protein during the labeling reaction. Upon addition of the NuLink reagent to a protein solution, NuLink reagents will react with nucleophilic residues on the protein to form a covalent bond in the form of an amide bond. After the reaction has been incubated and the protein purified by gel filtration, it is important to determine the DOL to know the amount of functionality that has been added to the sample. When using traditional reagents this determination is made using a secondary assay, such as the HABA assay which measures biotin incorporation when using biotinylation strategies such as NHS-biotin. Due to the spectroscopic properties of the NuLink reagents, it is possible to determine the amount of functionality that has been incorporated without the use of a secondary assay. The absorption spectrum can provide information about the relative amounts of components in solution using their spectroscopic properties. NuLink reagents have an absorbance at 330 nm which falls mostly outside the biological window, allowing for the use of UV/Vis spectroscopy on a UV transparent quartz cuvette for determination of the DOL. DOL = A ''( ε ''(*+,-+./ A 01( ε 01(2*3/+4. Equation 1: Uncorrected degree of labeling calculation A 330 Absorbance of the sample at 330 nm ε 330reagent Molar extinction coefficient (in M -1 cm -1 ) of the NuLink reagent at 330 nm A 280 Absorbance of the sample at 280 nm ε 280protein Molar extinction coefficient (in M -1 cm -1 ) of pure protein at 280 nm Table 1: Equation 1 variables

2 Molar extinction coefficients for protein must be experimentally determined by the user. This is typically performed using a simple rearrangement of Beer s Law on a standard laboratory spectrophotometer (Equation 2). A = ε c L Equation 2: Beer s law calculation of absorbance A Absorbance of the sample ε Molar extinction coefficient (in M -1 cm -1 ) c Concentration of the sample (M) L Pathlength of the cuvette (cm) L = 1 cm in standard spectrophotometers Table 2: Equation 2 and Equation 3 variables Reorganizing this equation after measuring a sample of precise concentration allows the user to accurately determine the molar extinction coefficient of a sample protein at any wavelength of interest (Equation 3). For use with NuLink reagents, extinction data of the protein at 280 nm is required for calculation of the degree of labeling. While performing this measurement it is also important to take the absorbance of the protein 330 nm to determine if there will be contribution from the protein to the absorbance at 330 nm, if so, a special calculation is required; see Proteins with contribution to absorbance at 330 nm below. Most standard laboratory spectrophotometers can be set to scan from 250 nm to 450 nm which will give all the information required for both calculations. Conversely, single point scans at both 280 nm and 330 nm will provide sufficient information for both calculations as well. ε = A c L Equation 3: Manipulated Beer s law calculation for experimental determination of the molar extinction coefficient (ε) of the pure protein.

3 Calculation of the degree of labeling provides insight to the average number of NuLink reagents that have been covalently incorporated onto a protein sample after the reaction has been purified. By taking the absorbance of the purified solution at 330 nm over the extinction coefficient of the reagent at 330 nm and dividing by the absorbance at 280 nm over the extinction coefficient of the pure protein at 280 nm (Equation 1) the DOL is obtained through the resultant molar ratio of reagent per protein. This represents the amount of functionality installed on the protein during the reaction. The DOL calculation requires the accurate measurement of the protein s absorbance at 280 nm. NuLink reagents have a contribution to the absorbance at 280 nm (Figure 1), for this reason it is important to use a correction factor (C 280 ) for the calculation of an accurate DOL (Equation 4). Absporbance (A.U.) BSA NuLink-BSA Wavelength (nm) Figure 1: Example data of absorbance before and after NuLink modification. Note the increased 280 nm absorbance from contribution of NuLink reagent. C 01( = A 01(*+,-+./ A ''(*+,-+./ Equation 4: Calculation of the correction factor for reagent. This data is obtained by NuChemie during molar extinction coefficient determination and is available online in the individual product specification sheets.

4 A 280reagent Absorbance of the reagent at 280 nm in extinction experiment A 330reagent Absorbance of the reagent at 330 nm in extinction experiment Table 3: Equation 4 variables Using C 280, the amount of absorbance the reagent has contributed to the overall absorbance at 280 nm can be determined through the measurement of the labeled sample at 330 nm. Since the correction factor is simply a ratio of absorbance at 280 nm per absorbance at 330 nm, the samples measured absorbance at 330 nm multiplied by the correction factor will give the expected amount of 280 nm absorbance by the NuLink reagent that has been conjugated to the sample. This contribution is subtracted from the overall absorbance at 280 nm and multiplied by the ε reagent which is measured at 330 nm. The final calculation of the DOL gives a unitless value for the average number of reagent molecules that are bonded to the sample protein with a correction for the reagents absorbance at 280 nm to prevent underestimation of the amount of reagent incorporated (Equation 5). DOL = A ''( ε 01(2*3/+4. A 01( A ''( C 01( ε ''(*+,-+./ Equation 5: Corrected degree of labeling formula A 330 Absorbance of the sample at 330 nm ε 280protein Molar extinction coefficient (in M -1 cm -1 ) of pure protein at 280 nm A 280 Absorbance of the sample at 280 nm C 280 Correction factor of reagent s contribution to absorbance at 280 nm ε 330reagent Molar extinction coefficient (in M -1 cm -1 ) of the NuLink reagent at 330 nm Table 4: Equation 5 variables

5 Proteins with contribution to absorbance at 330 nm. For most samples using equation 5 is sufficient to acquire an accurate DOL as the 330 nm absorbance of pure protein is typically negligible and arises from the presence of tryptophan residues. In special cases where the absorbance of the protein at 330 nm is not negligible a second correction factor is required. C 330 is utilized to accurately determine the reagents absorbance at 330 nm to prevent select samples from interfering with the measurement. C ''( = A ''(2*3/+4. A 01(2*3/+4. Equation 6: Correction factor for protein with absorbance at 330 nm A 280protein Absorbance of the protein at 280 nm in extinction experiment A 330protein Absorbance of the reagent at 330 nm in extinction experiment Table 5: Equation 6 variables C 330 can experimentally determined during the measurement of the extinction coefficient of the protein by taking the value of the absorbance at both 280 nm and 330 nm and inserting them into equation 6 above. This number will then be inserted into the DOL calculation much like the correction factor in equation 5 giving equation 7. DOL = (A ''( A 01( C ''( )ε 01(2*3/+4. A 01( A ''( C 01( ε ''(*+,-+./ Equation 7: Degree of labeling for protein with 330 nm contribution A 330 Absorbance of the sample at 330 nm ε 280protein Molar extinction coefficient (in M -1 cm -1 ) of pure protein at 280 nm A 280 Absorbance of the sample at 280 nm C 280 Correction factor of reagent s contribution to absorbance at 280 nm ε 330reagent Molar extinction coefficient (in M -1 cm -1 ) of the NuLink reagent at 330 nm C 330 Correction factor of protein s contribution at 330 nm Table 6: Equation 7 variables

6 Example calculations. This is an example calculation of the DOL for the BSA sample in figure 1 using equation 5. A ε 280protein M -1 cm -1 at 280 nm A C ε 330reagent 3725 M -1 cm -1 at 330 nm Table 7: Example data for degree of labeling calculation DOL = M DE cm DE M DE = 6.7 cmde Equation 8: Degree of labeling output for biotinylated BSA sample using equation 5 Values of C 280 and ε 330reagent for NuLink reagents are available online under the product specifications. Values for ε 280protein will be determined by the experimenter or obtained from protein supplier. A 330 and A 280 will be concentration dependent, samples of insufficient concentration will not be successful for the determination of the DOL. Additionally, measurements of low DOL require special considerations when labeling proteins due to the low extinction of the reagent when compared to most proteins it can be difficult to distinguish. Careful consideration of the measurement concentration should be made. Use of dual wavelength HPLC detector scanning UV at both 280 nm and 330 nm can alleviate some of these difficulties.

Lecture 5. More on UV-visible Spectrophotometry: Beer s Law and Measuring Protein Concentration

Lecture 5. More on UV-visible Spectrophotometry: Beer s Law and Measuring Protein Concentration Biological Chemistry Laboratory Biology 3515/Chemistry 3515 Spring 2018 Lecture 5 More on UV-visible Spectrophotometry: Beer s Law and Measuring Protein Concentration 23 January 2018 c David P. Goldenberg