254_Tryptophan quantification using UV fluorescence measurements on the CLARIOstar multi-mode microplate reader

Mark Gröne, Franka Maurer, BMG LABTECH, 07/2014
  • Tryptophan dilution curve shows linearity over a broad concentration range
  • Sensitivity was determined to be < 2 nM tryptophan
  • Combination of filters for excitation and LVF monochromatorTM for emission provides excellent UV fluorescence

Introduction

Tryptophan is one of the 22 amino acids of which proteins are built. It is an essential amino acid in the human diet as it cannot be synthesized by the human organism and has to be externally taken from food. Furthermore, tryptophan is used as a precursor substance for neurohormones, neuro-transmitters as well as for vitamins. 

Due to its essential nature, tryptophan is one of the most investigated amino acids. Originally, researchers aimed at identifying nutrients with high tryptophan content for dietary recommendations. Initially, tryptophan was determined by spectrophotometry (Fig. 1) as its aromatic residue enables detection by absorbance  (Fig. 2).

Fig. 1: Tryptophan absorbance spectrum recorded on the CLARIOstar using 1 nm resolution. In the MARS Data Analysis software the spectral curve was normalized to 100 % for the OD value at 280 nm.

Fig. 2: Structural formula of L-tryptophan. The blue marked part shows the aromatic residue.

With the further development of analytical instrumentation, researchers realized that fluorescence could be exploited for tryptophan determination. Moreover, it was observed that the position of tryptophan within the protein affected its fluorescence. In particular, if the aromatic tryptophan residue is present on the surface of the protein, the fluorescence is much higher compared to the fluorescence that can be captured when tryptophan is located inside the protein.  

Thanks to this observation, fluorescence could be used to determine the conformational state of a protein and its folding process. 

In this application note, we use the CLARIOstar multi-mode microplate reader from BMG LABTECH  for the measurement of tryptophan with fluorescence intensity detection. The CLARIOstar can use either filter or combine filters and LVF monochromatorsTM for accurate and sensitive detection of tryptophan.

Materials & Methods

  • L-tryptophan from SigmaAldrich
  • PBS from Biochrom
  • 96-well UV-star microplates from Greiner
  • CLARIOstar microplate reader from BMG LABTECH

Standard Curve Preparation
A 10 μM tryptophan stock solution was prepared in PBS buffer. From the stock, a 10 point dilution was prepared to contain the concentrations shown in Table 1. Pure PBS buffer served as blank. In every well of the 96-well plate, 300 μl of standard or blank was pipetted. Triplicates for every standard and 18 blanks were pipetted.

Table 1: Tryptophan dilution series

 

Content Concentration (M) Content Concentration (M)
S1 5.0E-6 S6 5.0E-8
S2 2.5E-6 S7 2.5E-8
S3 1.0E-6 S8 1.3E-8
S4 5.0E-7 S9 6.3E-9
S5 1.0E-7 S10 1.3E-9
    Blank 0

 

Instrument settings

 

Measurement method: Fluorescence Intensity, Endpoint mode
Number of flashes: 100
Setting time: 0.2

 

Optical settings

 

Using filter – LVF monochromator combination
excitation dichroic emission
F: 280-12 automatic monochromator monochromator 362-25
Using filters
excitation dichroic emission
F: 280-12 F: Trp-LP F: 360-20

 

Automated focus and gain adjustments were performed to obtain the highest dynamic range. The optimal focus for a fill volume of 300 μl resulted in 11.0 mm. Fixed gain and focus values were used for plate-to-plate comparisons with the same filling volume. 

Sensitivity (LOD) calculation 
The LOD was calculated according to IUPAC standards:

 

LOD = 3* SDblank/ slopestandard curve

 

Results & Discussion

Fig. 3: Tryptophan linearity range measurements using either a combination of filter and LVF monochromator or just filters. The insert zooms into the low nM concentration range.

The standard curve measurement showed a high linearity relation between tryptophan concentration and fluorescence output over a broad concentration range  (Fig. 3). 

The use either of an emission filter or of the emission LVF monochromator did not affect the sensitivity and accuracy of the measurement, demonstrating that the  LVF monochromator has filter-like sensitivity.

Conclusion

The data shown in this application note demonstrate that the CLARIOstar performs accurate and highly sensitive tryptophan fluorescence measurements. The limit of detection was determined to be < 2 nM. This corresponds to a tryptophan concentration of 0.4 ng/ml. This sensitivity can be achieved using either filter for both excitation and emission or with a filter for excitation and the LVF monochromator for emission.

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