Binding of prothrombin and its fragment 1 to phospholipid membranes studied by the solvent relaxation technique

Abstract

The phospholipid headgroup mobility of small unilamellar vesicles composed of different mixtures of phosphatidyl-L-serine (PS) and phosphatidylcholine is characterized by the solvent relaxation behavior of the polarity sensitive dyes 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and 6-palmitoyl-2-[trimethylammoniumethyl]-methylamino]naphthalene chloride (Patman). If the PS content exceeds 10%, the addition of calcium leads to a substantial deceleration of the solvent relaxation of both dyes, indicating the formation of Ca(PS)2 complexes. Addition of prothrombin and its fragment 1 leads to a further decrease of the headgroup mobility, as explained by the binding of more than two PS-molecules by a single protein molecule. Prodan monitors the outermost region of the bilayer and it clearly distinguishes between the binding of prothrombin and its fragment 1. The deeper incalated Patman does not distinguish between both proteins. The validity of the solvent relaxation technique for the investigation of the membrane binding of peripheral proteins is demonstrated by the studies of prothrombin induced changes in the steady-state fluorescence anisotropies of 1,6-diphenyl-1,3, 5-hexatriene.

Probing Ethanol-Induced Phospholipid Phase Transitions by the Polarity Sensitive Fluorescence Probes Prodan and Patman

“…a strong red shift to about 515 nm at high ethanol concentrations (Fig. This behaviour strongly suggests that no major changes in both polarity and viscosity of the dye environment take place, as it has been shown extensively that Patman is quite sensitive to any such change [22,23,28]. Thus, it might be assumed that again some kind of phase transition might occur similar to the DPPC bilayer system.…”

“…The validity of this approach has been demonstrated in several recent publications [22][23][24], and summarised in a few reviews [28,35,36] phase bilayers is very slow; thus, hardly any red shift with time is detectable (Fig. The validity of this approach has been demonstrated in several recent publications [22][23][24], and summarised in a few reviews [28,35,36] phase bilayers is very slow; thus, hardly any red shift with time is detectable (Fig.…”

“…Such dyes described in recent publications were based on 2-amino-substituted naphthalene [22,23] and anthracene-9carboxylic acid [24]. Such dyes described in recent publications were based on 2-amino-substituted naphthalene [22,23] and anthracene-9carboxylic acid [24].…”

“…Regarding the complex solvent relaxation (SR) behaviour of Prodan and Patman studied in detail during the last years, however [22,23,28], this conclusion might be an oversimplification. While the chromophore of Prodan is supposed to be located in the headgroup region of phospholipid bilayers close to the lipid/water interface the chromophore of Patman (similar to that of Laurdan) [29] has been shown to be embedded deeper in the bilayer, where it should be fixed tightly due to its long acyl chain [22].…”

Bilayer localization of membrane‐active peptides studied in biomimetic vesicles by visible and fluorescence spectroscopies

“…Recent studies have demonstrated that suitable fluorescent dyes located within either the hydrophilic headgroup region or the hydrophobic core of lipid bilayers facilitate direct observation of viscosity and polarity changes at the local environments of the probes [32–35]. Here we measured the effects of membrane peptides upon the SR of the fluorescent dye 6‐hexadecanoyl‐2‐(((2‐(trimethylammonium)ethyl)methyl)amino)‐naphthalene chloride (Patman) [33,36–38], incorporated in the vicinity of the glycerol interface within the phospholipid domains in the lipid/PDA vesicles (Fig. 1).…”