Fluorescent dyes modify properties of proteins used in microvascular research

Abstract

Objective: Fluorescent dyes, used frequently to label proteins for microvascular experiments, are assumed to not alter the protein's physicochemical characteristics. We tested the validity of that assumption for two probes, bovine serum albumin (BSA) and alpha-lactalbumin.

Methods: Standard electrophoretic techniques were used to examine the influence of five fluorescent dyes on the following three properties of BSA: molecular size (sodium dodecyl sulfate-polyacrylamide gel electrophoresis [PAGE]); relative molecular charge (native PAGE); and isoelectric point (pI) (i.e., isoelectric focusing). Also examined were the influence of the dyes on the relative charge of alpha-lactalbumin (native PAGE). Paired measures of single-vessel flux were made using two commercial preparations of dye-BSA on porcine coronary arterioles and venules.

Results: The addition of fluorescein isothiocyanate (FITC) to BSA altered significantly its size (p < 0.01; n = 6), relative charge (p < 0.02; n = 7), and pI. The other dyes caused minimal, yet significant, changes on the relative charge of BSA without influencing size or pI. Only FITC altered the relative charge of alpha-lactalbumin. While arteriolar fluxes of FITC-BSA and tetramethyl rhodamine isothiocyanate (TRITC)-BSA did not differ (p = 0.43; n = 5), in venules the FITC-BSA flux was greater than the TRITC-BSA flux (p < 0.001; n = 6).

Conclusions: The addition of fluorescent dyes, particularly FITC, alters the physicochemical characteristics of two widely used protein probes. These findings may affect the interpretation of microvascular permeability experiments performed using fluorescent dyes, and they suggest care in the selection of dyes for such experiments.