Analyzing the Interaction of Human Serum Albumin and 3,5-Diiodosalicylic Acid

Download PDF August 25, 2022


Human serum albumin (HSA) is the most abundant protein in blood plasma. HSA binds with pharmaceutical compounds and other in-vivo substances and plays an important role in the transport of these substances to target organs. Several studies have reported on the binding affinity of HSA and its interactions with a variety of compounds.

This application notes demonstrates the use of the J-1500 CD spectrometer and ATS-530 automatic titrator to monitor the interaction of HSA titrated with 3,5-diiodosalicylic acid. While 3,5-diiodosalicylic acid is achiral, its interaction with chiral HSA induces circular dichroism.

J-1500 Circular Dichroism Spectrophotometer
JASCO J-1500 CD spectrometer


Measurement Conditions 
 Data Acquisition Interval 2 sec
 Scan Speed 100 nm/min
 Spectral Bandwidth 1 nm
 Data Pitch 0.1 nm
 Path Length 10 mm
 Accumulations 2

50 mL aliquots of 0.025 mM of 3,5-diiodosalicylic acid was added to 2 mL of 0.0228 mM of HSA in 100 mM acetate buffer (pH 6.3) 20 times. The CD spectrum was scanned from 360 to 260 nm and also monitored at 320 nm.


J-1500, circular dichroism, HSA, pharmaceuticals, ATS-530 automatic titrator, biochemistry, pharmaceutical


Chirality can be induced in an achiral substance interacting with a chiral substance. This interaction will exhibit circular dichroism. The resulting CD spectrum from the interaction of achiral 3,5-diiodosalicylic acid with chiral HSA shows a positive peak at 320 nm in Figure 1. While HSA does not show a CD signal at 320 nm, the induced CD from the interaction of HSA and 3,5-diiodosalicylic acid depicts this signal at 320 nm which increases with the increasing additions of 3,5-diiodosalicylic acid and is shown in Figure 2.

Figure 1. 3-D graphical representation of the CD spectrum of HSA titrated with 3,5-diiodosalicylic acid.
Figure 2. CD spectra of HSA titrated with 3,5-diiodosalicylic acid. The arrows represent the increasing volume of 3,5-diiodosalicylic acid.

The increase in the induced CD signal at 320 nm was plotted in Figure 3 as a funtion of the 3,5-diiodosalicylic acid concentration. Figure 3 is also known as a Hill plot which describes the cooperativity of the interaction between HSA and 3,5-diiodosalicylic acid. A dissociation constant, which is the concentration at which 50% of a subtsance is bound, can also be estimated from a Hill plot. Using Figure 3, the dissociation constant of 3,5-diiodosalicylic acid binding to HSA was determined to be 0.023 mM. The Hill coefficient is approximately 3.1 which indicates a positive cooperative reaction.

Figure 3. CD signal monitored at 320 nm for the titration of 3,5-diiodosalicylic acid to HSA


This application note demonstrates the use of the ATS-530 automatic titrator and J-1500 CD spectrometer to obtain CD spectra of the titration of 3,5-diiodosalicylic to HSA in order to determine a dissociation constant.


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3. Sinha, S. S., Mitra, R. K., and S. K. Pal, J. Phys. Chem.B. (2008) 112, 4884-4891

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About the Author

Leah Pandiscia received her PhD from Drexel University where she studied Biophysical Chemistry. She is a Spectroscopy Applications Scientist.