TY - JOUR

T1 - Acridine orange as a probe for measuring pH gradients across membranes

T2 - Mechanism and limitations

AU - Palmgren, Michael Gjedde

PY - 1991/2/1

Y1 - 1991/2/1

N2 - Acridine orange is an optical probe commonly used to monitor pH gradients across membranes. In the present study, the changes observed in the visible absorption spectrum of acridine orange during intravesicular acidification of oat root plasma membrane vesicles are shown to be identical with those obtained by increasing the free dye concentration, adding anions, or lowering the temperature, but different from those obtained on addition of biological membranes. It is therefore suggested that the absorbance changes observed during the formation of the pH gradient are simply due to accumulation of free dye inside the vesicles and subsequent dimerization, and not the result of dye-membrane interactions. The proportion of monomeric acridine orange that could undergo dimerization decreased with decreasing temperature. Furthermore, in a membrane-free system different anions induced the formation of dimer-excimer complexes to different degrees. During the formation of the pH gradient permeant anions present in the reaction medium follow the movement of protons into the vesicles, and the intravesicular accumulation of anions thereby amplifies acridine orange quenching, the degree of amplification being dependent on the anion species. Therefore, the use of acridine orange, and probably all metachromatic dyes, as probes for monitoring pH gradients is limited, since these probes neither reflect quantitatively the amount of H+ pumped nor the effect of anions and temperature on transmembrane H+ transport.

AB - Acridine orange is an optical probe commonly used to monitor pH gradients across membranes. In the present study, the changes observed in the visible absorption spectrum of acridine orange during intravesicular acidification of oat root plasma membrane vesicles are shown to be identical with those obtained by increasing the free dye concentration, adding anions, or lowering the temperature, but different from those obtained on addition of biological membranes. It is therefore suggested that the absorbance changes observed during the formation of the pH gradient are simply due to accumulation of free dye inside the vesicles and subsequent dimerization, and not the result of dye-membrane interactions. The proportion of monomeric acridine orange that could undergo dimerization decreased with decreasing temperature. Furthermore, in a membrane-free system different anions induced the formation of dimer-excimer complexes to different degrees. During the formation of the pH gradient permeant anions present in the reaction medium follow the movement of protons into the vesicles, and the intravesicular accumulation of anions thereby amplifies acridine orange quenching, the degree of amplification being dependent on the anion species. Therefore, the use of acridine orange, and probably all metachromatic dyes, as probes for monitoring pH gradients is limited, since these probes neither reflect quantitatively the amount of H+ pumped nor the effect of anions and temperature on transmembrane H+ transport.

UR - http://www.scopus.com/inward/record.url?scp=0026069460&partnerID=8YFLogxK

U2 - 10.1016/0003-2697(91)90542-2

DO - 10.1016/0003-2697(91)90542-2

M3 - Journal article

C2 - 1827963

AN - SCOPUS:0026069460

VL - 192

SP - 316

EP - 321

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 2

ER -