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Implications of Volume Exclusion: A Look at Thermodynamic Perspective of DNA-Hemoglobin Complexes and Their Reconstitutes Under Macromolecular Crowding

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Abstract

Live cells contain high concentrations of macromolecules, but almost all experimental biochemical data have been generated from dilute solutions that do not reflect conditions in vivo. To understand biomolecular behavior in vivo, properties studied in vitro are extrapolated to conditions in vivo. Another significant factor which is overlooked is the effects of macromolecular crowding and its consequences in the actual biochemical and physiological environment. Such influences of crowding, its modification and physiological parameters have been reported. The present study investigates the effect of molecular crowding on binding characteristics of Salmon sperm DNA with Bovine hemoglobin and their reconstitutes in presence of molecular crowders viz., Poly ethylene glycol (PEG) and Dextran of different molecular weight by fluorescence, UV visible spectroscopic technique at different temperatures. The results showed that BHb fluorescence was quenched by sDNA through static quenching mechanism which is enhanced in presence of polymers. The number of binding sites ‘n’ and binding constants ‘K’ were determined at different temperatures based on fluorescence quenching. The thermodynamic parameters namely ∆H°, ∆G°, T∆S° were studied at different temperatures and the results indicate that hydrophobic forces are predominant in the sDNA-BHb complex. Negative ∆G° values imply that the binding process is spontaneous.

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Abbreviations

sDNA:

Salmon sperm deoxyribose nucleic acid

BHb:

Bovine hemoglobin

PEG:

Poly ethylene glycol

PBS:

Phosphate buffered saline

EtBr:

Ethidium bromide

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Acknowledgments

We gratefully acknowledge the Department of Science and Technology (DST), Government of India, for their financial support through the DST INSPIRE - AORC scheme.

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Authors and Affiliations

  1. Department of Biophysics, University of Mumbai, Vidyanagri Santacruz (E), Mumbai, 400098, India

    Priyanka D. Pal, Prabhakar M. Dongre & Arunkumar V. Chitre

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  1. Priyanka D. Pal
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  2. Prabhakar M. Dongre
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  3. Arunkumar V. Chitre
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Correspondence to Arunkumar V. Chitre.

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Pal, P.D., Dongre, P.M. & Chitre, A.V. Implications of Volume Exclusion: A Look at Thermodynamic Perspective of DNA-Hemoglobin Complexes and Their Reconstitutes Under Macromolecular Crowding. J Fluoresc 26, 355–362 (2016). https://doi.org/10.1007/s10895-015-1721-z

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  • DOI: https://doi.org/10.1007/s10895-015-1721-z

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