Conformation and activity dependent interaction of glucose oxidase with CdTe quantum dots: towards developing a nanoparticle based enzymatic assay.
Nanomolar concentrations of cysteine-capped CdTe quantum dots (QDs) quenched the fluorescence of tryptophan moieties in glucose oxidase (GOX), while the fluorescence of the other fluorophore in the enzyme, FAD (Flavin Adenine Dinucleotide), was not affected by the QDs. The quenching followed a linear Stern-Volmer equation and its static nature was confirmed by time-resolved photoluminescence (PL) spectroscopy. The binding of substrate to the GOX resulted in a decrease of the Stern-Volmer quenching constant, which is attributable to a change in tertiary structure of GOX as revealed by circular dichroism (CD) spectroscopy. The quenching constant was further lowered for the free tryptophan. A strong size dependence of quenching pattern was observed and the quenching efficiency increased with increasing average size of the CdTe QDs. For a given size, quenching constants exhibit an increasing trend with a gradual decrease in polarity of the solvent indicating binding between GOX and CdTe QDs. Fourier-transform infra-red (FTIR) spectroscopy has revealed the involvement of the indole ring of tryptophan in binding with CdTe QDs. Interestingly, the quenching pattern also showed a strong dependence on activity of the enzyme. An empirical equation has been derived to correlate the enzyme activity with the quenching constant based on which a novel QD-based fluorimetric method for activity determination could be devised.
Priyam
Amiya
A
UGC-DAE Consortium for Scientific Research, Kolkata Centre, III/LB-8, Bidhannagar, Kolkata 700098, India.
Chatterjee
Anindita
A
Bhattacharya
Subhash C
SC
Saha
Abhijit
A
D001681:Biological Assay; D002104:Cadmium / Q000737:chemistry*; D002942:Circular Dichroism; D003545:Cysteine / Q000378:metabolism; D005949:Glucose Oxidase / Q000378:metabolism*; D053758:Nanoparticles / Q000737:chemistry*; D011487:Protein Conformation; D045663:Quantum Dots; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D013691:Tellurium / Q000737:chemistry*; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
D002104:Cadmium; D014364:Tryptophan; D005949:Glucose Oxidase; D003545:Cysteine; D013691:Tellurium