Article pubs.acs.org/Langmuir
Human Fibrinogen Monolayers on Latex Particles: Role of Ionic Strength Anna Bratek-Skicki,† Paulina Ż eliszewska,† Zbigniew Adamczyk,*,† and Michał Cieśla‡ †
J. Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Science, Niezapominajek 8, 30-239 Cracow, Poland M. Smoluchowski Instutute of Physics, Jagiellonian University, Reymonta 4, 30-059 Cracow, Poland
‡
ABSTRACT: The adsorption of human serum fibrinogen on polystyrene latex particles was studied using the microelectrophoretic and concentration depletion methods. Measurements were carried out for pH 3.5 and an ionic strength range of 10−3 to 0.15 M NaCl. The electrophoretic mobility of latex was determined as a function of the amount of adsorbed fibrinogen (surface concentration). A monotonic increase in the electrophoretic mobility (zeta potential) of the latex was observed, indicating a significant adsorption of fibrinogen on latex for all ionic strengths. No changes in the latex mobility were observed for prolonged time periods, suggesting the irreversibility of fibrinogen adsorption. The maximum coverage of fibrinogen on latex particles was precisely determined using the depletion method. The residual protein concentration after making contact with latex particles was determined by electrokinetic measurements and AFM imaging where the surface coverage of fibrinogen on mica was quantitatively determined. The maximum fibrinogen coverage increased monotonically with ionic strength from 1.8 mg m−2 for 10−3 M NaCl to 3.6 mg m−2 for 0.15 M NaCl. The increase in the maximum coverage was interpreted in terms of the reduced electrostatic repulsion among adsorbed fibrinogen molecules. The experimental data agree with theoretical simulations made by assuming a 3D unoriented adsorption of fibrinogen. The stability of fibrinogen monolayers on latex was also determined in ionic strength cycling experiments. It was revealed that cyclic variations in NaCl concentration between 10−3 and 0.15 M induced no changes in the latex electrophoretic mobility, suggesting that there were no irreversible molecule orientation changes in the monolayers. On the basis of these experimental data, a robust procedure of preparing fibrinogen monolayers on latex particles of well-controlled coverage was proposed.
1. INTRODUCTION Fibrinogen (Fb) is the one of the most abundant blood plasma proteins,1 playing an essential role in the clotting cascade, platelet adhesion, leucocyte binding, thrombosis, angiogenesis, inflammatory response, tumor growth, and fouling of artificial organs.2−6 Therefore, a knowledge of fibrinogen adsorption mechanisms at solid/electrolyte interfaces is of great practical significance. This provoked much research aimed at determining its properties in the bulk and interactions with various surfaces. The shape and dimensions of fibrinogen were determined by the electron microscopy studies of Hall and Slayter,7 who established that this molecule has a colinear, trinodular conformation with a total length of 47.5 nm. The two equal end domains are spherical in shape and have a diameter of 6.5 nm; the middle domain has a diameter of 5 nm. These domains were connected by cylindrical rods having a diameter of 1.5 nm. These fibrinogen dimensions and the molecule shape were confirmed by numerous studies carried out using atomic force microscopy (AFM).8−11 The primary structure (amino acid sequence) of fibrinogen was determined in refs 12 and 13. In was shown that the molecule is a symmetric dimer composed of three identical pairs of polypeptide chains, referred to traditionally as the Aα, Bβ, and γ chains. They are coupled in the middle of the molecule through a few disulfide bridges forming a central © 2013 American Chemical Society
nodule. The longest chain (Aα) is composed of 610 amino acids, the Bβ chain comprises 460 amino acids, and the γ chain is composed of 411 amino acids. Accordingly, from this chemical structure, the molar mass of the fibrinogen molecule is predicted to be 337 897 D.13 Additionally, by analyzing the chemical structure one can conclude that major parts of the Aα chains extend from the core of the molecule, which forms two appendages each having a molar mass equal to 42 300 D. These fragments of polypeptide Aα chains are collapsed under dry conditions that can be seen in the crystallographic structure of fibrinogen (Table 1). However, in electrolyte solutions they assume more expanded conformations depending on the ionic strength and pH. This plays an essential role in the hydrodynamic behavior of the molecule as was shown in ref 14. The most expanded conformation is predicted for pH
