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

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

By Heather Haffner

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January 5, 2024

Introduction

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

Experimental

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.

Keywords

200-CD-0014, J-1500, Circular Dichroism, CD, HSA, pharmaceuticals, ATS-530 automatic titrator, biochemistry, pharmaceutical

Results

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

Conclusion

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.

References

1. R. Brodersen, J. Biol. Chem. (1977) 252, 14, 5067-5072.
2. U. Kragh-Hansen, Biochem. J. (1983) 209, 135-142.
3. Sinha, S. S., Mitra, R. K., and S. K. Pal, J. Phys. Chem.B. (2008) 112, 4884-4891

This document has been prepared based on information available at the time of publication and is subject to revision without notice. Although the contents are checked with the utmost care, we do not guarantee their accuracy or completeness. JASCO Corporation assumes no responsibility or liability for any loss or damage incurred as a result of the use of any information contained in this document. Copyright and other intellectual property rights in this document remain the property of JASCO Corporation. Please do not attempt to copy, modify, redistribute, or sell etc. in whole or in part without prior written permission.

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About the Author

JASCO Application Note

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

Introduction

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

Experimental

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.

Results

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

Conclusion

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.

Keywords

200-CD-0014, J-1500, Circular Dichroism, CD, HSA, pharmaceuticals, ATS-530 automatic titrator, biochemistry, pharmaceutical

References

1. R. Brodersen, J. Biol. Chem. (1977) 252, 14, 5067-5072.
2. U. Kragh-Hansen, Biochem. J. (1983) 209, 135-142.
3. Sinha, S. S., Mitra, R. K., and S. K. Pal, J. Phys. Chem.B. (2008) 112, 4884-4891

This document has been prepared based on information available at the time of publication and is subject to revision without notice. Although the contents are checked with the utmost care, we do not guarantee their accuracy or completeness. JASCO Corporation assumes no responsibility or liability for any loss or damage incurred as a result of the use of any information contained in this document. Copyright and other intellectual property rights in this document remain the property of JASCO Corporation. Please do not attempt to copy, modify, redistribute, or sell etc. in whole or in part without prior written permission.
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