TY - JOUR
T1 - A Mechanistic Investigation of Methylene Blue and Heparin Interactions and Their Photoacoustic Enhancement
AU - Wang, Junxin
AU - Jeevarathinam, Ananthakrishnan Soundaram
AU - Humphries, Kathryn
AU - Jhunjhunwala, Anamik
AU - Chen, Fang
AU - Hariri, Ali
AU - Miller, Bill R.
AU - Jokerst, Jesse V.
N1 - Publisher Copyright:
Copyright © 2018 American Chemical Society.
PY - 2018/11/21
Y1 - 2018/11/21
N2 - We recently reported a real-time method to measure heparin in human whole blood based on the photoacoustic change of methylene blue (MB). Intriguingly, the MB behaved unlike other "turn on" photoacoustic probes - the absorbance decreased as the photoacoustic signal increased. The underlying mechanism was not clear and motivated this study. We studied the binding mechanism of MB and heparin in water and phosphate buffer saline (PBS) with both experimental and computational methods. We found that the photoacoustic enhancement of the MB-heparin mixture was a result of MB-heparin aggregation due to charge neutralization and resulting sequestration of MB in these aggregates. The sequestration of MB in the MB-heparin aggregates led to decreased absorbance - there was simply less free dye in solution to absorb light. The highest photoacoustic signal and aggregation occurred when the number of negatively charged sulfate groups on heparin was approximately equal to the number of positively charged MB molecule. The MB-heparin aggregates dissociated when there were more sulfated groups from heparin than MB molecules because of the electrostatic repulsion between negatively charged sulfate groups. PBS facilitated MB dimer formation regardless of heparin concentration and reprecipitated free MB in aggregates due to ionic strength and ionic shielding. Further molecular dynamics experiments found that binding of heparin occurred at the sulfates and glucosamines in heparin. Phosphate ions could interact with the heparin via sodium ions to impair the MB-heparin binding. Finally, our model found 3.7-fold more MB dimerization upon addition of heparin in MB solution confirming that heparin facilitates MB aggregation. We conclude that the addition of heparin in MB decreases the absorbance of the sample because of MB-heparin aggregation leading to fewer MB molecules in solution; however, the aggregation also increases the PA intensity because the MB molecules in the MB-heparin aggregate have reduced degrees of freedom and poor heat transfer to solvent.
AB - We recently reported a real-time method to measure heparin in human whole blood based on the photoacoustic change of methylene blue (MB). Intriguingly, the MB behaved unlike other "turn on" photoacoustic probes - the absorbance decreased as the photoacoustic signal increased. The underlying mechanism was not clear and motivated this study. We studied the binding mechanism of MB and heparin in water and phosphate buffer saline (PBS) with both experimental and computational methods. We found that the photoacoustic enhancement of the MB-heparin mixture was a result of MB-heparin aggregation due to charge neutralization and resulting sequestration of MB in these aggregates. The sequestration of MB in the MB-heparin aggregates led to decreased absorbance - there was simply less free dye in solution to absorb light. The highest photoacoustic signal and aggregation occurred when the number of negatively charged sulfate groups on heparin was approximately equal to the number of positively charged MB molecule. The MB-heparin aggregates dissociated when there were more sulfated groups from heparin than MB molecules because of the electrostatic repulsion between negatively charged sulfate groups. PBS facilitated MB dimer formation regardless of heparin concentration and reprecipitated free MB in aggregates due to ionic strength and ionic shielding. Further molecular dynamics experiments found that binding of heparin occurred at the sulfates and glucosamines in heparin. Phosphate ions could interact with the heparin via sodium ions to impair the MB-heparin binding. Finally, our model found 3.7-fold more MB dimerization upon addition of heparin in MB solution confirming that heparin facilitates MB aggregation. We conclude that the addition of heparin in MB decreases the absorbance of the sample because of MB-heparin aggregation leading to fewer MB molecules in solution; however, the aggregation also increases the PA intensity because the MB molecules in the MB-heparin aggregate have reduced degrees of freedom and poor heat transfer to solvent.
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U2 - 10.1021/acs.bioconjchem.8b00639
DO - 10.1021/acs.bioconjchem.8b00639
M3 - Article
C2 - 30281976
AN - SCOPUS:85055125111
SN - 1043-1802
VL - 29
SP - 3768
EP - 3775
JO - Bioconjugate Chemistry
JF - Bioconjugate Chemistry
IS - 11
ER -