Empirical equation for the correction of fluorescence quenching of proteinaceous substance by Suwannee river natural organic matter

Abstract

Fluorescence quenching of proteinaceous substances by natural organic matter is a well-known phenomenon, but there are no known methods for correcting it. The main objective of this research was to develop an empirical equation to correct the fluorescence quenching of different concentrations of bovine serum albumin (BSA – 0.15, 0.25, 0.5, 0.75, 1, 1.25 μmol/L (μM)) by Suwannee river natural organic matter (SWNOM - 0,2,4,6,8,10 mg-C/L) using the fluorescence titration method. The excitation emission matrix (EEM) data were analyzed by parallel factor analysis with inner filter effect removal. With increasing SWNOM concentration, BSA peak intensity quenching was in the range 29–85%, with a linear relationship for increment of either BSA or SWNOM concentration. A higher ratio of SWNOM to BSA resulted in greater BSA peak intensity quenching. The unquenched BSA peak (BSA (RU)) is given by the empirical equation. BSA (RU) = {2.052 × peak C (RU) + 2.522} × quenched peak T (RU)0.624 The calculated unquenched BSA peak intensities using the empirical equation agreed well with the actual unquenched peak values (R2 = 0.98, mean absolute error = 0.33 RU). The equation is expected to help in rapid estimation of the quenching effect of SWNOM on BSA. Highlights Empirical equation to correct the fluorescence quenching of bovine serum albumin by Suwannee river natural organic matter is discussed.; The EEM data was analyzed by parallel factor analysis with inner filter effect removal.; With an increase in SWNOM concentration, quenching of BSA peak intensity was in the range 29–85%.; Higher ratio of SWNOM to BSA resulted in higher quenching of the peak intensity of BSA.;

Keywords

• bovine serum albumin
• dissolved organic matter
• fluorescence titration
• parafac
• protein-humic substance complex
• excitation emission matrix