Determining metal biding affinity of hProCA32 using Tb 3+ EGTA/DTPA

(A) The fluorescence spectra of hProCA32-Ca2+ titration using metal chelator buffer

system with excitation at 280 nm and emission range of 300-400 nm. (B) The fitted curve of the maximum intensity at 311 nm with a dissociation constant of 1.35 × 10-8 M using hill equation (Equation 5-2).

5.2.4. Determining metal biding affinity of hProCA32 using Tb3+ EGTA/DTPA buffer system

Lanthanides tend to bind to EF-hand calcium-binding proteins with high affinity.

Terbium (Tb3+)is a trivalent lanthanide possessing similar ionic radius and coordination as

calcium. Due to its unique fluorescence properties, Tb3+ is frequently used as a probe in

fluorescence spectroscopy to study the intrinsic metal binding properties of biomolecules. In

FRET, accessible tryptophan upon excitation transfers energy to terbium and generates

fluorescence signal. Tryptophan is excited at a wavelength of 282 nm, which emits at 340 nm; a

it emits the energy between 545 nm and 595 nm. Our previously developed rat ProCA32 has

been reported to have a strong binding affinity for Tb3+ of about 10-22 M. The humanized

ProCA32 must have a similar binding affinity to Tb3+. However, it is extremely difficult to

accurately measure the binding affinity in such small range using fluorescence. Fluorescence

titration has limited sensitivity and the free metal in the solution can generate its own

fluorescence. In order to overcome these limitations, metal chelator buffer system can be used to

control the free metal in the system. The dissociation constants for chelators such as EGTA and

DTPA to Tb3+ are obtained from the National Institute of Standards and Technology (NIST).

Changes in Trp fluorescence can be monitored to analyze the metal binding affinities at

extremely low concentrations of free Tb3+. The Tryptophan (Trp) residue present near the

binding site can be excited at 280 nm, which has an emission at 315 nm. This can further excite

the Tb3+ that is bound to the protein, and the fluorescence emission can be observed at 545 nm.

This system contains 50 mM HEPES, 150 mM NaCl, 30 μM hProCA32, 5mM EGTA or DTPA and 0.1 μL Rhod-5N, pH7.2. EGTA and DTPA have a Kdto Tb3+

of 10-18 M and 9.55 ×

10-22 M, respectively. The system contains about 170 times higher concentration of the chelator

than that of the protein; therefore the majority of the protein should be in its apo form (metal free

form). Tb-EGTA system can create a buffer range from 10-18 to 10-9 M of free Tb3+. EGTA is a

weak chelator as compared to DTPA and can be used Tb-DTPA can generate a buffer range

between 10-22 and 10-18 M of free Tb3+. The Kd of the protein is first tested in the weaker buffer

range using EGTA followed by, the stronger buffer range using DTPA. As a weak metal binding

indicator, the fluorescence of Rhod-5N can increase when free [Tb3+] is higher than 10-6 M. Tb3+

itself has its own fluorescence, thus as a background control, a parallel experiment was

buffer and Tb3+ was titrated to the system.

Figure 5.6 A and Figure 5.7 B are the control experiments with Buffer- 5mM EGTA and

buffer- 5mM DTPA, respectively. Various volumes of Tb 3+ were titrated into the system. B and

D are the titration plots with 30 µM of hProCA32 into buffer-EGTA and Buffer-DTPA,

respectively with fluorescence intensity subtracted from the background Tb3+ from the control

experiment. Various volumes of Tb 3+ were titrated in to the system, as the [Tb3+] increased the

fluorescence intensity at 545 nm, which is an indication of Tb3+ binding to hProCa32.

The increase in fluorescence intensities at 545 nm in Figure 5.6 and Figure 5.7

demonstrates that energy transfer between Tb3+ and hProCA32 occurred in both Tb-EGTA and

Tb-DTPA buffer systems. Therefore, hProCA32 can bind to Tb3+ ata low concentration range of

10-18 to 10-22 M. The fluorescence intensities at 545 nm were plotted using hill curve fitting, and

binding affinity constants were calculated using Hill equation. hProCA32 has the Kd_{hCA32,Tb of}

1.41x10-18 M using Tb-EGTA buffer system and KdhCA32,Tb of 7.79 x 10 -22

M using Tb-DTPA

Figure 5.6 Fluorescence spectra of hProCA32 binding with Tb3+ using metal chelator buffer system.

(A)The EGTA buffer system contained 50 mM HEPES, 150 mM NaCl at pH7.2, and 5 mM EGTA was used a control where(B) contained 50 mM HEPES, 150 mM NaCl at pH7.2, 5

mM EGTA and 30 μM hProCA32. As the Tb 3+

was titrated in to the system, the fluorescence intensity at 545 increased due to the binding of protein to the free metal. Once the total metal concentration is higher than the DTPA, low affinity dye Rhod-5N binds to the remaining metal causing an increase in fluorescence intensity at 575 nm, which indicates that the system has reached its saturation point. The curve has fluorescence intensities subtracted with the backgroundsignal generated by Tb3+ itself. The curve fitting of these intensities at 545 were done using Hillequation. (C) The actual fluorescence intensities plotted to monitor the dynamic range and (D) the normalized fluorescence intensities to obtain the binding affinities.

Figure 5.7 Determining Tb 3+binding affinity of hProCA32 using Tb3+-EGTA / DTPA system

(A) The EGTA buffer system contains 50 mM HEPES, 150 mM NaCl at pH7.2, 5 mM EGTA and 30 μM hProCA32. (C) Saturation test where the maximum fluorescence

intensity peak corresponds to Rhod-5N-Tb3+ complex where the minimum fluorescence