Researchers have developed a series of dye-affinity chromatographic stationary phases by immobilizing reactive dyes onto collagen fibers derived from cattle hides to achieve selective separation and purification of lysozyme. Column chromatography serves as a widely applied method for protein downstream processing based on differential solute-matrix interactions, but conventional stationary phases relying on porous particles frequently encounter elevated back pressure and limited ligand utilization as particle sizes decrease. To overcome these constraints, fibrous stationary phases offer lower mass transfer resistance and rapid flow capabilities, making natural collagen fibers an attractive structural scaffold due to their hierarchical network structure, non-toxic nature, and abundant sidechain carboxylic, hydroxyl, amino, and amide groups. By covalently attaching reactive dye molecules that mimic natural biological conformations onto these collagen matrices, the research team engineered an affinity adsorbent system that demonstrates high static and dynamic adsorption capacities alongside selectivity during target protein extraction.

The investigation evaluated six distinct reactive dyes spanning azo, anthraquinone, and phthalocyanine chromogens, including reactive red 118, reactive blue 171, reactive blue 19, reactive blue 114, reactive blue 21, and reactive blue 116, using lysozyme as a bioactive model protein. Pristine collagen fibers without functional ligands exhibited poor adsorption performance due to insufficient binding sites and weak interaction forces. When modified with reactive dyes, the adsorption capacity of the resulting stationary phases correlated closely with the specific chemical structure and functional groups of the immobilized ligands. Anthraquinone dyes demonstrated superior performance compared to azo and phthalocyanine counterparts, with reactive blue 114 functionalized fibers achieving the highest lysozyme adsorption capacity at 1000.30 milligrams per gram, closely followed by reactive blue 19. In contrast, phthalocyanine dye matrices exhibited limited capacity because their bulky and rigid macrocyclic frameworks hindered effective contact with the target protein, while azo dyes relied primarily on surface electrostatic interactions.

Synthesis of the dye-affinity stationary phases was performed by suspending cattle hide collagen fibers in a dye solution containing sodium chloride, stirring the mixture at 40 degrees Celsius for four hours, and subsequently introducing sodium carbonate for an additional two hours of continuous reaction. After filtering, washing sequentially with deionized water and ethanol until the filtrate was dye-free, and drying at 50 degrees Celsius, the modified fibers underwent comprehensive physical and chemical characterization. Scanning electron microscopy and energy-dispersive X-ray spectroscopy mapping confirmed that the regular D-periodicity observed on pristine collagen disappeared, yielding a rougher surface uniformly covered with sulfur from the grafted dye molecules. For the optimal reactive blue 114 modified fibers, elemental analysis revealed an increase in sulfur content reaching 2.88 percent, while visible fibrillar structures remained intact to facilitate mass transfer. Fourier transform infrared spectroscopy showed characteristic hydroxyl stretching at 3435 and 3287 inverse centimeters, an amide band at 1635 inverse centimeters, and a newly emerged peak at 1030 inverse centimeters corresponding to sodium sulfonate functional groups, which was further confirmed by X-ray photoelectron spectroscopy.

Batch adsorption investigations revealed that solution pH exerts a pronounced impact on lysozyme uptake due to the amphoteric nature of protein surfaces. Zeta potential measurements demonstrated that the reactive blue 114 functionalized fibers maintained negative surface charges across a pH range from 4.0 to 11.0, with negativity intensifying at higher alkalinity. Because lysozyme possesses an isoelectric point of 10.7, the protein carries a net positive charge in acidic and neutral conditions, establishing strong electrostatic attraction with the negatively charged fiber stationary phase between pH 4.0 and 10.0. The measured adsorption capacity progressively increased as pH rose from 4.0 to 8.0, achieving an optimal static capacity of 1536.92 milligrams per gram at pH 8.0. Even under alkaline conditions at pH 11.0, where both the adsorbent matrix and the protein exhibited net negative charges, significant adsorption was preserved, confirming that retention does not depend exclusively on electrostatic interactions but involves multiple cooperative binding mechanisms.

Selective adsorption capabilities were examined using binary protein solutions combining lysozyme with either bovine serum albumin or ovalbumin at pH 8.0 with initial protein concentrations of 1.0 milligram per milliliter. The functionalized fibers demonstrated extremely low adsorption toward bovine serum albumin at 151.38 milligrams per gram and ovalbumin at 172.85 milligrams per gram, yielding separation factors of 96.56 for lysozyme over bovine serum albumin and 87.59 for lysozyme over ovalbumin. In these mixed systems, lysozyme uptake remained robust at approximately 870 milligrams per gram, with minor reductions attributed to competitive occupancy of limited sites via weak electrostatic interactions. Evaluation of elution conditions established that 1.0 molar sodium chloride or 1.0 molar ammonium sulfate at pH 8.0 recovered only 65.23 percent and 53.19 percent of adsorbed protein, whereas an elution buffer of 10 millimolar phosphate containing 1.0 molar sodium chloride and 20 percent ethanol achieved a 97.01 percent recovery yield. In repeated cycling using this solvent mixture, the fibrous matrix retained over 80 percent of initial capacity across three cycles.

Kinetic profiling of lysozyme uptake on the reactive blue 114 stationary phase at 25, 30, and 35 degrees Celsius demonstrated rapid initial adsorption during the first hour followed by a gradual stabilization toward dynamic equilibrium within four hours. Higher operating temperatures accelerated mass transfer and shortened the duration required to reach equilibrium, reflecting the accessible surface sites provided by the open fibrous architecture. Applying pseudo-first-order and pseudo-second-order models revealed that the pseudo-second-order kinetic model yielded consistently higher correlation coefficients across all temperatures, indicating that surface chemisorption functions as the rate-limiting step. Equilibrium isotherm analysis between 0.8 and 3.0 milligrams per milliliter at pH 8.0 showed saturation around 1.8 milligrams per milliliter, with experimental maximum capacities increasing from 1594.23 milligrams per gram at 25 degrees Celsius to 1701.51 at 30 degrees Celsius and 1939.13 at 35 degrees Celsius. The Langmuir model exhibited superior fitting relative to the Freundlich formulation, generating theoretical maximum capacities of 1586.06, 1699.21, and 1960.66 milligrams per gram, demonstrating monolayer adsorption and an endothermic process.

Molecular docking calculations conducted with Maestro and visualized using PyMol provided atomic-scale insight into the interaction geometry between reactive dye ligands and lysozyme crystal structures. Specifically, reactive blue 114 formed hydrophobic interactions with phenylalanine 34, tryptophan 62, tryptophan 63, isoleucine 98, alanine 107, valine 109, and alanine 110. Concurrently, the sulfonate groups of reactive blue 114 established electrostatic contacts with glutamic acid 35, aspartic acid 48, aspartic acid 52, arginine 61, and arginine 114, while hydrogen bonds formed with threonine 47, aspartic acid 48, and arginine 114. The pyrimidine active moiety further supported hydrogen bonding, demonstrating how aromatic rings, active heterocycles, and sulfonate substituents cooperate to drive ligand recognition.

Fluorescence quenching experiments and disruptive agent titrations substantiated the multi-force adsorption model governing the interaction between reactive blue 114 and lysozyme. Fluorescence spectra demonstrated ground-state complex formation, pinpointing tryptophan residues as the primary interaction locus, while double logarithmic analysis yielded binding constants exceeding 10,000 liters per mole at all tested temperatures. Thermodynamic calculations revealed negative Gibbs free energy values reflecting spontaneous binding, accompanied by positive enthalpy changes of 150.40 kilojoules per mole and positive entropy changes of 619.43 joules per mole-kelvin. This thermodynamic signature indicates that endothermic conformational adaptations expose hydrophobic binding domains while liberating structured water molecules into bulk solution. Disruptive agent tests demonstrated that increasing sodium chloride from zero to 1.0 molar reduced lysozyme uptake from 1428.11 to 610.83 milligrams per gram, confirming electrostatic attraction as the primary driver. Adding 20 percent isopropanol under 1.0 molar salt dropped capacity to 150.67 milligrams per gram, whereas urea caused minimal reduction, delineating a force hierarchy dominated by electrostatics, followed by hydrophobic effects and hydrogen bonding.

Continuous-flow dynamic adsorption was evaluated by packing 1.0 gram of the functionalized fibers into a column measuring 20.0 centimeters in length and 1.0 centimeter in internal diameter, feeding a 1.0 milligram per milliliter lysozyme feed at pH 8.0. At a volumetric flow rate of 0.25 milliliters per minute, the column sustained 103.60 bed volumes, processing 1220.5 milliliters of solution prior to reaching the breakthrough point at 4882 minutes. Doubling the flow rate to 0.50 milliliters per minute lowered the breakthrough time to 2063 minutes and shortened the mass transfer zone, with Thomas model kinetic constants shifting from 1.23 times 10 to the negative sixth to 1.95 times 10 to the negative sixth liters per milligram-minute due to reduced contact time. Nevertheless, theoretical dynamic adsorption capacities calculated from the Thomas model remained exceptionally resilient, declining by merely 5.4 percent from 1504.28 milligrams per gram at 0.25 milliliters per minute to 1423.77 milligrams per gram at 0.50 milliliters per minute, demonstrating excellent convective transport through the fibrous matrix.

The practical separation efficiency of the functionalized fiber column was confirmed through stepwise chromatographic fractionation of a binary ovalbumin and lysozyme mixture followed by direct extraction from raw biological feedstocks. When loading a 10.0 milligram per milliliter binary protein mixture at pH 8.0, unadsorbed ovalbumin eluted rapidly with neutral buffer, while lysozyme was firmly retained until displaced by an elution buffer composed of 1.2 molar sodium chloride and 20 percent isopropanol, achieving a chromatographic resolution of 0.66 with recovery efficiencies of 99.35 percent for ovalbumin and 82.57 percent for lysozyme. To evaluate real-world utility, fresh chicken egg white was diluted fourfold and centrifuged to isolate the supernatant containing native lysozyme at approximately 3.5 percent total protein content. High-performance size-exclusion chromatography verified that the characteristic lysozyme retention peak at 13.87 minutes completely vanished from the egg white supernatant upon fiber contact and was subsequently recovered with high purity in the eluate, underscoring the potential of collagen fiber affinity matrices for industrial protein downstream processing.

Subject of Research: Dye-affinity collagen fiber stationary phases for protein chromatography

Article Title: Dye-ligand functionalized collagen fibers as an affinity chromatography stationary phase for efficient protein separation

Article References: Cheng, J., Li, Z., Pang, N., Luo, W., & Liao, X. (2026). Dye-ligand functionalized collagen fibers as an affinity chromatography stationary phase for efficient protein separation. Collagen and Leather, 8(1), Article 44. https://doi.org/10.1186/s42825-026-00269-2

Image Credits: AI Generated

DOI: 10.1186/s42825-026-00269-2

Keywords: affinity chromatography, collagen fibers, protein purification, lysozyme, reactive dyes, adsorption capacity, downstream bioseparation, mass transfer, Dye-ligand, functionalized, collagen, fibers