Adsorption Of Lactoferrin Research Articles

Adsorption of lactoferrin and bovine serum albumin nanoparticles on pellicular two-layer agarose-nickel at reactive blue 4 in affinity chromatography

In this study, adsorption of lactoferrin (Lf) and bovine serum albumin (BSA) nanoparticles on pellicular two-layer agarose-nickel immobilized by reactive blue 4 dye-ligand (2L-AN@RB4) was investigated. 2L-AN@RB4 was prepared using the three-phase emulsion method. The dynamic light scattering was first employed to quantify size distribution of Lf and BSA nanoparticles. Then, scanning electron microscopy (SEM) and atomic force microscope (AFM), respectively, were used to characterize structures of the two types of nanoparticles and adsorbent beads. SEM and AFM images demonstrate that shapes of the nanoparticles are globular and relatively uniform, where no adhesions were observed. Finally, adsorption behaviors of both Lf and BSA nanoparticles on 2L-AN@RB4 in affinity chromatography were investigated, with focus on the adsorption kinetics. Influences of contact time, pH, and initial concentration were analyzed to investigate the adsorbent behaviors in the expanded bed column. The influence of contact time on adsorption of Lf versus BSA nanoparticles indicates that 4 h is enough to adsorb protein models equal to 45%. Results indicate that Lf nanoparticles have a higher rate of around 83% than that of BSA nanoparticles. The increases in pH have negative effect on adsorption while opposite trend was found for initial concentration. Adsorption isotherm results reveal that the Langmuir and Freundlich isotherm models fit both adsorption kinetics of Lf and BSA very well.

Analysis of the Calcium Phosphate-Based Hybrid Layer Formed on a Ti-6Al-7Nb Alloy to Enhance the Ossseointegration Process.

This paper reports on hybrid, bioactive ceramic Ca-P-based coating formation on a Ti-6Al-7Nb alloy substrate to enhance the osseointegration process. The Ti alloy was anodized in a Ca3(PO4)2 suspension and then the additional layer was formed by the sol-gel technique to obtain a mixture of the calcium phosphate compounds. The oxide layer was porous and additional ceramic particles were formed after sol-gel treatment (scanning electron microscopy analysis coupled with energy-dispersive x-ray spectroscopy). The ceramic particles were formed on some parts of the oxide layer and did not completely fill the pores. The layer thickness of the anodized Ti alloy was comprised between 3.01 and 5.03 µm and increased to 7.52–12.30 µm after the formation of an additional layer. Post-treatment of the anodized Ti alloys caused a decrease in surface roughness, and the layer became strongly hydrophilic. Crystalline phase analysis (X-ray diffraction, XRD) showed that the hybrid layer was composed of TiO2 (anatase), Ca3(PO4)2, Ca10(PO4)6(OH)2 and a partially amorphous phase; thus, the layer was also analyzed by Raman spectroscopy. The hybrid layer showed worse adhesion to the substrate than the anodized layer only; however, the coating was not brittle, and the first delamination of the layer was determined at 1.84 ± 0.11 N during scratch-test measurement. The hybrid coating was favorable for collagen type I and lactoferrin adsorption, strongly influencing the proliferation of osteoblast-like MG-63 cells. The coatings were cytocompatible and may find applications in formation of the functional layers on long-term implants’ surface after.

Synthesis and characterization of chitosan mini-spheres with immobilized dye as affinity ligand for the purification of lactoperoxidase and lactoferrin from dairy whey

Lactoperoxidase (LP) and lactoferrin (LF) are two interesting proteins present in dairy whey due to their antioxidant, anti-inflammatory, antibacterial, antiviral and antifungal properties. LP and LF are traditionally purified using cation-exchange chromatography, but since the concentration of these proteins in sweet whey are very low (0.03–0.06 g/L for LP and around 0.08–0.20 g/L for LF), clarification, diafiltration, concentration and/or depletion of others whey proteins prior to the ion-exchange chromatography is needed to obtain good adsorption yields. After their elution from the matrix, the overall process results in variable yields. Due to their similar physico-chemical properties, a high cross-contamination is usually present, especially for the LP fraction, where part of the LF is co-eluted.The development of novel supports with immobilized affinity and/or multimodal ligands aims to the direct adsorption of the target proteins even at low concentration and without the need of a pre-treatment of the source. Herein, a new, low-cost support material using chitosan mini-spheres with Orange R-HE triazine dye as immobilized ligand (CMS-Orange R-HE) was synthesized, physico-mechanically characterized and used for the purification of LP and LF by a one-step direct adsorption from dairy whey and a two-step elution process, with minor cross-contamination. CMS-Orange R-HE matrix showed a compression modulus of 47.6 ± 9.8 kPa, predominantly elastic deformation and an open-porous structure with mesopores around 5–20 nm and without ink-bottle pores, ideal for protein purification applications. The affinity between LP and LF for CMS-Orange R-HE matrix was characterized using the Langmuir isotherm model (Kd = 0.48 ± 0.07 mg/mL and 0.31 ± 0.09 mg/mL; qmax = 77.5 ± 4.0 mg/g and 56.0 ± 2.1 mg/g, respectively). Adsorption studies showed that LP interaction with CMS-Orange R-HE was partially influenced by the presence of ionic strength (p < 0.05), whereas LF adsorption was not. These differences of both electrostatic and hydrophobic interactions between LP and LF with the CMS-Orange R-HE matrix were used to develop a differential elution process using Response Surface Methodology (RSM). By one-step direct adsorption from dairy whey and two differential elution steps –by using the CMS-Orange R-HE matrix- LP and LF were obtained with good yields (≈70% for LP and ≈60% for LF), high purity and with minor cross-contamination between them. The strategy presented here, could be applied in other cases of cross-contamination between two proteins, being especially interesting for its application in multimodal chromatography.

Targeting cancer with lactoferrin nanoparticles: recent advances.

Lactoferrin, an iron storage protein, is known for its microbicidal activity and its ability to modulate the immune system, mediated through specific interactions with receptors on cell surfaces for internalization. These activities confer a significant versatility to lactoferrin, presenting it as a targeting ligand to disease-bearing cells. Early efforts in developing targeted delivery systems have focused on nano- and microcomposites comprised of metal and polymeric materials. These can be targeted through conjugation or adsorption of lactoferrin to achieve recognition to receptor-expressing cells. More recently, efforts are underway to utilize lactoferrin itself as a medium in loading the therapeutic agent. The functional efficiency of drug-loaded lactoferrin nanoparticles has been evaluated in different disease conditions such as cancer, HIV, Parkinson's disease, etc. This review will present the details of composition and performance of various delivery systems designed and developed using lactoferrin as targeting agent for the treatment of cancer.

Lactoferrin adsorption onto silver nanoparticle interface: Implications of corona on protein conformation, nanoparticle cytotoxicity and the formulation adjuvanticity

Nanoparticle, in biological milieu, tends to bind a variety of biomolecules, especially protein, forming an interfacial corona that provides biological identity to nanoparticle. Understanding the protein corona is likely to provide insight in nanoparticle bio-distribution, nanoparticle-mediated cytotoxicity and potential nanoparticle-mediated applications. Thus, the bovine lactoferrin (BLf) corona onto silver nanoparticle (AgNP) interface is studied with reference to BLf conformational stability and the resulting complex’s cytotoxic and adjuvant properties. The adsorption of BLf onto AgNP interface is reflected by change in the particle-specific surface plasmon resonance (SPR). Additionally, the molecular dynamics (MD) simulation deciphered the binding energy and molecular recognition in BLf and AgNP complex. Protein adsorption onto the interface is majorly driven by van der Waals interactions and hydrogen bonds, without any significant effect on the protein conformation and stability. BLf retains bacteriostatic property in the conjugate, and the presence of BLf corona ameliorated the particle-mediated cytotoxicity, despite the enhanced cellular internalization. Additionally, BLf adsorbed AgNP formulation shows better adjuvanticity than AgNP only or BLf only formulation, as demonstrated using Bacillus anthracis protective antigen protein, in vivo.

STERIC MASS ACTION MODEL FOR LACTOFERRIN ADSORPTION IN CRYOGEL WITH IMMOBILIZED COPPER IONS

Parameters of equilibrium adsorption obtained from experiments using immobilized metal affinity chromatography (IMAC) were used to evaluate the applicability of the steric mass-action (SMA) model to describe the adsorption of lactoferrin to cryogel resin under different conditions. The adsorption of lactoferrin on continuous supermacroporous cryogel with immobilized Cu2+ ions was evaluated in batch adsorption experiments at different pH (6-8) and temperature (293-313 K) values. Estimated values of the equilibrium constant (K) and characteristic number of binding sites (n) showed that these parameters decreased with increasing ionic strength, pH and temperature, while the nonlinear parameter, the steric factor (σ), increased with increasing ionic strength and temperature. Expressions correlating these parameters with pH, ionic strength and temperature were then derived.

Adsorption Analysis of Lactoferrin to Titanium, Stainless Steel, Zirconia, and Polymethyl Methacrylate Using the Quartz Crystal Microbalance Method.

It is postulated that biofilm formation in the oral cavity causes some oral diseases. Lactoferrin is an antibacterial protein in saliva and an important defense factor against biofilm development. We analyzed the adsorbed amount of lactoferrin and the dissociation constant (K d) of lactoferrin to the surface of different dental materials using an equilibrium analysis technique in a 27 MHz quartz crystal microbalance (QCM) measurement. Four different materials, titanium (Ti), stainless steel (SUS), zirconia (ZrO2) and polymethyl methacrylate (PMMA), were evaluated. These materials were coated onto QCM sensors and the surfaces characterized by atomic force microscopic observation, measurements of surface roughness, contact angles of water, and zeta potential. QCM measurements revealed that Ti and SUS showed a greater amount of lactoferrin adsorption than ZrO2 and PMMA. Surface roughness and zeta potential influenced the lactoferrin adsorption. On the contrary, the K d value analysis indicated that the adsorbed lactoferrin bound less tightly to the Ti and SUS surfaces than to the ZrO2 and PMMA surfaces. The hydrophobic interaction between lactoferrin and ZrO2 and PMMA is presumed to participate in better binding of lactoferrin to ZrO2 and PMMA surfaces. It was revealed that lactoferrin adsorption behavior was influenced by the characteristics of the material surface.

Interfacial structures of whey protein isolate (WPI) and lactoferrin on hydrophobic surfaces in a model system monitored by quartz crystal microbalance with dissipation (QCM-D) and their formation on nanoemulsions

In this work, the interactions between two oppositely charged proteins in solution, namely whey protein isolate (WPI) and lactoferrin, were investigated for uses in nanoemulsions. ζ-potential and turbidity measurements indicated that these two proteins interact strongly via electrostatic attraction at pH 6. Quartz crystal microbalance with dissipation (QCM-D) was employed to study the adsorption behaviour and the formation of different interfacial structures of the two proteins onto a model hydrophobic surface that mimics the interface of an oil droplet in emulsions. Sequential addition of WPI and lactoferrin (or vice versa) formed thin and rigid protein bi-layers (8–10 nm). However, a viscous and thick surface layer (101 nm) was formed by the protein complex using a mixture of WPI and lactoferrin. High pressure homogenization and solvent evaporation were then used to replicate the QCM-D results on real nanoemulsion systems. Sequential adsorption of WPI and lactoferrin created bi-layered nanoemulsions with stable and relatively small droplets (90 nm in diameter). In contrast, the sequential adsorption of lactoferrin and WPI as well as the use of a mixture of WPI and lactoferrin (mixed layer) resulted in large aggregates with poor stability. This study provided insights into the stability of nanoemulsions influenced by structural features of interfacial layers based on two oppositely charged protein molecules.

Temperature-Switchable Agglomeration of Magnetic Particles Designed for Continuous Separation Processes in Biotechnology.

The purpose of this work was the synthesis and characterization of thermally switchable magnetic particles for use in biotechnological applications such as protein purification and enzymatic conversions. Reversible addition-fragmentation chain-transfer polymerization was employed to synthesize poly(N-isopropylacrylamide) brushes via a "graft-from" approach on the surface of magnetic microparticles. The resulting particles were characterized by infrared spectroscopy and thermogravimetric analysis and their temperature-dependent agglomeration behavior was assessed. The influence of several factors on particle agglomeration (pH, temperature, salt type, and particle concentration) was evaluated. The results showed that a low pH value (pH 3-4), a kosmotropic salt (ammonium sulfate), and a high particle concentration (4 g/L) resulted in improved agglomeration at elevated temperature (40 °C). Recycling of particles and reversibility of the temperature-switchable agglomeration were successfully demonstrated for ten heating-cooling cycles. Additionally, enhanced magnetic separation was observed for the modified particles. Ionic monomers were integrated into the polymer chain to create end-group functionalized particles as well as two- and three-block copolymer particles for protein binding. The adsorption of lactoferrin, bovine serum albumin, and lysozyme to these ion exchange particles was evaluated and showed a binding capacity of up to 135 mg/g. The dual-responsive particles combined magnetic and thermoresponsive properties for switchable agglomeration, easy separability, and efficient protein adsorption.

Targeted chemotherapy of visceral leishmaniasis by lactoferrin-appended amphotericin B-loaded nanoreservoir: in vitro and in vivo studies.

Exploitation of lactoferrin-appended amphotericin B bearing nanoreservoir (LcfPGNP-AmB) for targeted eradication of Leishmania donovani. LcfPGNP-AmB was architechtured through ionic adsorption of lactoferrin over core poly (d,l-lactide-co-glycolide) nanoparticles and characterized. Anti-Leishmania activity in visceral leishmaniasis models, immunomodulatory potential, biodistribution and toxicity profile were also assessed. LcfPGNP-AmB (size, 196.0 ± 5.28 nm; zeta-potential, +21.7 ± 1.52 mV; encapsulation efficiency, ∼89%) showed reduced toxicity, increased protective proinflammatory mediators expression and down-regulation of disease-promoting cytokines. Biodistribution study illustrated preferential accumulation of LcfPGNP-AmB in liver and spleen. LcfPGNP-AmB showed augmented antileishmanial activity by significantly reducing (∼88%) splenic parasite burden of infected hamsters, compared with commercial-formulations. Superior efficacy, desired stability and reliable safety of cost-effective LcfPGNP-AmB, suggest its potential for leishmaniasis therapeutics.

Inhibition of initial bacterial adhesion on titanium surfaces by lactoferrin coating.

Because dental implant abutments are located at transmucosal sites, their surface should inhibit bacterial accumulation to prevent peri-implantitis. The authors examined the effects of human lactoferrin (LF), an antibacterial protein present in saliva, as an antibacterial coating on the titanium surface and evaluated its effects before and after mucin-containing artificial saliva (AS) incubation. In the control group, titanium disks were soaked in distilled water, whereas in the LF group, titanium disks were soaked in LF solution to coat the disks. In the control-AS and LF-AS groups, half of the control and LF disks were incubated with AS. To confirm LF adsorption, the fluorescence intensity of fluorescein isothiocyanate-labeled LF was measured. The LF and LF-AS groups showed significantly higher intensity than the control and control-AS groups (P < 0.01). There was no significant difference between the LF and LF-AS groups (P > 0.05). The amount of adhered Streptococcus gordonii significantly increased by incubation with AS (P < 0.01) and significantly decreased by adsorption of LF (P < 0.01). There was no interaction between the two factors, LF adsorption and AS incubation (P = 0.561). These results suggest that the adsorbed LF inhibited bacterial adhesion following AS incubation. According to qualitative LIVE/DEAD analysis, viable bacteria appeared to be decreased in the presence of LF and SEM observation indicated that altered morphologies increased in LF and LF-AS groups. These results suggest that the adsorbed LF remained on the titanium surface after incubation with AS, and the remaining LF inhibited bacterial adhesion and exhibited bactericidal effects. Therefore, the adsorption of LF on the abutment material appears to be effective in preventing peri-implantitis.

Interaction of Whey Lactoferrin with Copper Immobilized in Polyacrylamide Cryogel

Design and optimization of Immobilized Metal Affinity Chromatography (IMAC) processes require deep knowledge of driving factors responsible for interaction between immobilized metal and biomolecules. Based on this requirement, interactions between lactoferrin from cheese whey and IDA-Cu2+-cryogel system was investigated. Data from adsorption of lactoferrin in the system at pH 6, 7 and 8, as well as NaCl concentration from 200 to 1000 mmol L−1 were adjusted Langmuir, Freundlich, Temkin and Langmuir-Freundlich isotherm models. Although all models were able to explain the interaction lactoferrin-cryogel system, the Langmuir-Freundlich model was the most accurate one. In addition, it could explain quantitatively the cooperativity and heterogeneity of the bounds between protein and matrix. The methods used in this project are useful for both better understanding of the protein-immobilized metal interactions and developing preparative scale IMAC.

Microcalorimetric study of the adsorption of lactoferrin in supermacroporous continuous cryogel with immobilized Cu2+ ions

The adsorption affinity of lactoferrin from whey in monolithic supermacroporous cryogel was analyzed using equilibrium data adsorptive isothermal titration microcalorimetry to measure thermodynamic information governing the process. Isotherm data was obtained at temperatures of 20, 30 and 40°C, pH 6, 7 and 8, and ionic strength of 200, 600 and 1000mmolL−1 NaCl. The Langmuir model was fitted to equilibrium data. The binding was tighter at higher temperatures. The adsorption of protein was observed as spontaneous in all cases analyzed. The microcalorimetric study indicated that, in most cases examined, the adsorption of the protein in the matrix was entropy and enthalpy favored and entropy driven. Results provide data to enable the improvement of technical processes for the affinity separation of proteins.

Direct binding of Concanvalin A onto iron oxide nanoparticles for fast magnetic selective separation of lactoferrin

Concanavalin A (Con A) was successfully bound onto Fe3O4 magnetic nanoparticles (MNPs) via carbodiimide activation as a magnetic nano-adsorbent for glycoprotein separation. The resulting Con A-bound Fe3O4 MNPs were nearly superparamagnetic with a mean diameter of 11.74±3.86nm. The size and structure of Fe3O4 MNPs had no significant changes after Con A binding. The binding of Con A on the surface of Fe3O4 MNPs was confirmed by the analysis of Fourier transform infrared spectroscopy. From the thermogravimetric analysis and the concentration change of Con A in the binding solution, the weight percentage of Con A in the typical Con A-bound Fe3O4 MNPs was determined to be about 10wt%. Furthermore, the Con A-bound Fe3O4 MNPs were found to be quite efficient for the fast adsorption of lactoferrin. The equilibrium was achieved within 5min and the adsorption data for lactoferrin obeyed the Langmuir isotherm equation. At pH 7 and 25°C, the maximum adsorption capacity and equilibrium constant were 59.2mg/g and 0.0103L/mg, respectively. In addition, by SDS–polyacrylamide gel electrophoresis, the capability of Con A-bound Fe3O4 MNPs for the selective adsorption of lactoferrin was further confirmed.

One-Step Purification of Lactoferrin from Crude Sweet Whey Using Cation-Exchange Expanded Bed Adsorption

In order to purify lactoferrin from crude sweet whey in one step, an expanded bed adsorption process was proposed with cation-exchanger Fastline SP. A series of experiments were carried out to assess the possibility of this process and optimize the operating conditions. First the adsorption equilibrium and kinetics of lactoferrin with Fastline SP were measured. The results indicated that Fastline SP had a fast adsorption of lactoferrin and was suitable for high-velocity operation. Then the expansion properties, liquid mixing, and breakthrough behaviors in the expanded bed were investigated, and the expansion factor was set at 2.0. In addition, the elution condition was optimized as 0.5 M NaCl in the phosphate buffer (pH 7.0). Finally, lactoferrin was successfully recovered with a high purity of 88.5% and a reasonable recovery of 77.1% in a single step. The purification factor reached 553.

Lactoferrin binding to transglutaminase cross-linked casein micelles

Casein micelles in skim milk were either untreated (untreated milk) or were cross-linked using transglutaminase (TGA-milk). Added lactoferrin (LF) bound to the casein micelles and followed Langmuir adsorption isotherms. The adsorption level was the same in both milks and decreased the micellar zeta potential, indicating charge neutralization and the formation of complex coacervate-type interactions. For the untreated milks, the adsorption of LF was initially accompanied by an increase in turbidity of the milk and in the size of the casein micelles; however, after several hours, the turbidity and the casein micelle size of these milks decreased markedly. For the TGA-milks, no change in casein micelle size was observed on adsorption of LF, but the turbidity increased due to the increased mass of the casein micelles, and remained constant on holding. These results indicate that the cross-linking of the casein micelles prior to adding LF prevents disintegration of the casein micelles.

Interfacial adsorption and denaturization of human milk and recombinant rice lactoferrin

Lactoferrin (LF) produced from recombinant technologies can achieve almost identical amino acid sequences and three-dimensional structures to those extracted from mammals, but differences often arise in the carbohydrate chains attached through N-glycosylation, with altered sizes, structures, and chemical nature. We compare the differences in solvation and interfacial adsorption from two samples, human milk lactoferrin (hLF) and recombinant rice lactoferrin (rLF). Lactoferrin is a bilobal protein with a molecular weight of about 80 kD. It has three N-glycosylation sites. Each of the three attached glycan chains on rLF contains seven to eight sugar groups. In comparison, each of the three glycan chains attached to hLF contains 12-13 sugar groups and is twice as long. The rLF meting point in 1 mg/ml aqueous solution (pH 7 phosphate buffer, I=20 mM) was 43 degrees C from dynamic light scattering, compared to 53 degrees C for hLF, exhibiting the enhanced solvation and stability of hLF due to its longer carbohydrate side chains. Silicon oxide surfaces provided a model substrate for assessment of lactoferrin adsorption and comparison with other proteins. The time dependent interfacial adsorption studied by spectroscopic ellipsometry (SE) was characterized by a fast initial step followed by a slow relaxation process. In addition, the SE results revealed the persistently higher adsorption of rLF, again showing the effect of glycan side chains. In spite of the different adsorbed amounts, neutron reflection revealed similar interfacial structures of the adsorbed protein layers. At the low lactoferrin concentration around 10 mg/l, a flat-on molecular monolayer formed with both LF lobes attached to the SiO(2) surface through electrostatic attraction. As the protein concentration increased, a secondary molecular layer further adsorbed to the first one and the attachment was again driven by electrostatic attraction. The intermixing between the globular lobes resulted in the dense packing in the middle 60 A with some of the lobes projected toward the aqueous bulk solution.

Patterning of lactoferrin using functional SAMs of iron complexes

A new method has been developed that allows spatially resolved adsorption of lactoferrin on a surface, by means of specific non-covalent interaction between the native protein and a patterned self-assembled monolayer of an iron-containing terpyridine complex.

Surface-Induced Unfolding of Human Lactoferrin

We have determined the structural conformations of human lactoferrin adsorbed at the air/water interface by neutron reflectivity (NR) and its solution structure by small angle neutron scattering (SANS). The neutron reflectivity measurements revealed a strong structural unfolding of the molecule when adsorbed at the interface from a pH 7 phosphate buffer solution (PBS with a total ionic strength at 4.5 mM) over a wide concentration range. Two distinct regions, a top dense layer of 15-20 angstroms on the air side and a bottom diffuse layer of some 50 angstroms into the aqueous subphase, characterized the unfolded interfacial layer. At a concentration around 1 g dm(-3), close to the physiological concentration of lactoferrin in biological fluids, the adsorbed amount was 5.5 x 10(-8) mol m(-2) in the absence of NaCl, but the addition of 0.3 M NaCl reduced protein adsorption to 3.5 x 10(-8) mol m(-2). Although the polypeptide distributions at the interface remained similar, quantitative analysis showed that the addition of NaCl reduced the layer thickness. Parallel measurements of lactoferrin adsorption in D2O instead of null reflecting water confirmed the unfolded structure at the interface. Furthermore, the D2O data indicated that the polypeptide in the top layer was predominantly protruded out of water, consistent with it being hydrophobic. In contrast, the scattering intensity profiles from SANS were well described by a cylindrical model with a diameter of 47 angstroms and a length of 105 angstroms in the presence of 0.3 M NaCl, indicating a retention of the globular framework in the bulk solution. In the absence of NaCl but with the same amount of phosphate buffer, the length of the cylinder increased to some 190 angstroms and the diameter remained constant. The length increase is indicative of changes in distance and orientation between the bilobal monomers due to the change in charge interactions. The results thus demonstrate that the surface structural unfolding was caused by the exposure of the protein molecule to the unsymmetrical energetic balance following surface adsorption.

Interactions between adsorbed lactoferrin layers measured directly with the atomic force microscope

Lactoferrin is an important protein found in tear fluid and has been implicated in the fouling of contact lenses. The interaction forces between lactoferrin layers, adsorbed onto hydrophobic model substrates, as a function of electrolyte concentration, solution pH and protein concentration were investigated using an atomic force microscope (AFM) where the geometry of the scanning tip was modified by attaching a colloid particle. Bovine lactoferrin adsorbed strongly and modified the interaction forces to include both a long-range electrostatic repulsive force, which could be described using the DLVO theory, and a short-range additional repulsion due to compression of the adsorbed protein layers. The adsorption of lactoferrin was verified by XPS analysis of the surfaces and the adsorbed layer was determined to be present as a full monolayer by AFM imaging. At smaller separation distances, deviations from theoretical predictions indicated that there was an adsorbed layer of at least 5 nm on both surfaces. DLVO theory could be used successfully to describe the longer ranged interaction forces in background electrolyte concentrations of 0.0002 and 0.001 M KNO 3, at various solution pH values. This analysis yielded effective diffuse electrical double layer potentials, which when plotted against the solution pH, suggested that the point of zero charge (pzc) of the protein coated surfaces was around pH 8, in agreement with literature values of the pI for lactoferrin. At higher electrolyte concentrations, 0.15 M KNO 3, the interaction forces could no longer be described using DLVO theory. The interaction forces between the adsorbed lactoferrin layers were purely repulsive, and the range and magnitude varied as a function of solution pH. Variations in the forces as a function of solution pH reflected conformational changes that depend on short ranged electrostatic interactions between the peptide residues within the protein molecules and the peptide residues and the substrate as well as van der Waals and hydrophobic interactions. The range of the interaction forces and the high compressibility of the adsorbed layers indicated that a significant number were present in an end-on orientation.