Size Exclusion Chromatography Analysis Research Articles

In-solution behavior and protective potential of asparagine synthetase A from Trypanosoma cruzi

l-Asparagine synthetase (AS) acts in asparagine formation and can be classified into two families: AS-A or AS-B. AS-A is mainly found in prokaryotes and can synthetize asparagine from ammonia. Distinct from other eukaryotes, Trypanosoma cruzi produces an AS-A. AS-A from Trypanosoma cruzi (Tc-AS-A) differs from prokaryotic AS-A due to its ability to catalyze asparagine synthesis using both glutamine and ammonia as nitrogen sources. Regarding these peculiarities, this work uses several biophysical techniques to provide data concerning the Tc-AS-A in-solution behavior. Tc-AS-A was produced as a recombinant and purified by three chromatography steps. Circular dichroism, dynamic light scattering, and analytical size exclusion chromatography showed that Tc-AS-A has the same fold and quaternary arrangement of prokaryotic AS-A. Despite the tendency of protein to aggregate, stable dimers were obtained when solubilization occurred at pH ≤ 7.0. We also demonstrate the protective efficacy against T. cruzi infection in mice immunized with Tc-AS-A. Our results indicate that immunization with Tc-AS-A might confer partial protection to infective forms of T. cruzi in this particular model.

Functionalized Artificial Bidomain Proteins Based on an α-Solenoid Protein Repeat Scaffold: A New Class of Artificial Diels–Alderases

αRep is a family of entirely artificial repeat proteins. Within the previously described αRep library, some variants are homodimers displaying interdomain cavities. Taking advantage of these properties, one of these homodimers called αRep A3 was converted into entirely artificial single chain bidomain metalloenzymes. A nonmutated A3 domain was covalently linked with an A3′ domain bearing a unique cysteine on a chosen mutated position (F119C or Y26C). This single mutation ensured the covalent coupling of a 1:1 copper(II)/phenanthroline or copper(II)/terpyridine complex as a catalytic center within the interdomain cavity which was maintained large enough to accommodate two substrates of the Diels–Alder (D–A) reaction. This allowed us to obtain four new artificial Diels–Alderases that were fully characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, UV–vis spectroscopy, and size exclusion chromatography analyses and were then further used for the catalysis of the D–A r...

Novel concrete superplasticizers based on phosphate esters

Novel polyphosphate superplasticizers were synthesized by copolymerization reaction between 2-(methacryloyl oxy) ethyl phosphate monomer and the macromonomer polyethylene glycol methacrylate ester at different molar ratios.The synthesized phosphated comb polymers were characterized by size exclusion chromatography and elemental analysis and their dispersing performance in cement was assessed via mini slump tests in cement paste.The phosphated comb polymers exhibited superior dispersing performance over conventional polycarboxylate comb polymers (PCEs), were less retarding on cement and showed comparable robustness against sulfate and clay impurities. These properties can be explained by the high calcium complexing capacity of the phosphate groups. Thus, phosphated comb polymers present a viable alternative to polycarboxylate superplasticizers.

Study on Speciation of As, Cr, and Sb in Bottled Flavored Drinking Water Samples Using Advanced Analytical Techniques IEC/SEC-HPLC/ICP-DRC-MS and ESI-MS/MS.

The main aim of the research was to develop a complementary analytical approach consisting of bespoke speciation analysis and non-targeted speciation analysis of As, Sb, and Cr in flavored bottled drinking water samples using HPLC/ICP-DRC-MS and ESI-MS/MS. The scope of two previously developed analytical procedures, (1) multielemental speciation procedure for AsIII, AsV, CrVI, SbIII, and SbV analysis and (2) arsenic speciation procedure for AsB, AsIII, DMA, MMA, and AsV quantification, was extended to the analysis of a new sample type in terms of bespoke speciation analysis. As for the non-targeted speciation, analysis size exclusion chromatography was used with ICP-MS and a complementary technique, ESI-MS/MS, was used for the organic species of As, Sb, and Cr screening. Full validation of procedures 1 and 2 was conducted. Procedure 1 and 2 were characterized with precision values in the range from 2.5% to 5.5% and from 3.6% to 7.2%, respectively. Obtained recoveries ranged from 97% to 106% and from 99% to 106% for procedures 1 and 2, respectively. Expanded uncertainties calculated for procedures 1 and 2 ranged from 6.1% to 9.4% and from 7.4% to 9.9%, respectively. The applicability of the proposed procedures was tested on bottled drinking water samples. Results for the real samples in procedure 1 were in the range from 0.286 ± 0.027 [μg L−1] to 0.414 ± 0.039 [μg L−1] for AsIII, from 0.900 ± 0.083 [μg L−1] to 3.26 ± 0.30 [μg L−1] for AsV, and from 0.201 ± 0.012 [μg L−1] to 0.524 ± 0.032 [μg L−1] for SbV. CrVI and SbIII were not detected in any sample. As for procedure 2, results were in the range from 0.0541 ± 0.0053 [μg L−1] to 0.554 ± 0.054 [μg L−1] for AsB. Results for AsIII and AsV obtained with procedure 2 were in good accordance with results obtained with procedure 1. DMA and MMA were not detected in any sample.

Characterization of native-like HIV-1 gp140 glycoprotein expressed in insect cells

The trimeric HIV-1 envelope glycoprotein (Env) is critical for vaccine development aimed at achieving broadly-neutralizing antibody responses. The use of various recombinant expression systems and construct designs are associated with the resultant nature of produced proteins, especially in terms of glycosylation, antigenicity, and immunogenicity of the glycoprotein. Here, we explored an otherwise baculovirus cassette than classical one designed to express HIV-1 Env protein, including SOSIP mutation and Foldon moiety involvement. This improved design increased the ratio of the Env trimer fraction from ∼40% to ∼60% with respect to that of prototypical design, as indicated by high-performance size-exclusion chromatography and sedimentation velocity analysis. In addition, the design prolonged cell viability and enhanced the final yield (approximately 13–15 mg/L) after affinity purification. gp140 produced from insect cells mimicked the native-like trimer and mainly adopted glycosylation pattern of oligomannose glycans. The native-like Env proteins conferred cross-clade neutralizing antibody production in BALB/c mice. In summary, the expression of Env in insect cells by optimizing the baculovirus vector provides an alternative strategy for HIV-1 immunogen production and may benefit future Env-based HIV vaccine design.

Effects of formulation development methods on the stability of model protein pharmaceuticals embedded in solid lipid matrices

This study was conducted to investigate the influence of formulation development methods on the stability (secondary structure, aggregation, and biological activity) of protein drugs embedded in lipid matrices. Catalase, horseradish peroxidase, and α-chymotrypsin were employed as model proteins, while Precirol® AT05 (glyceryl palmitostearate) was used as lipid matrix. Protein-loaded lipid matrices were prepared using melting and mixing and wet granulation methods. Attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy, size exclusion chromatography (SEC) and biological activity analyses were performed. ATR FT-IR analysis indicated significant interference of the lipid with the protein amide-I band, which was eliminated using spectral subtraction. Wet granulation method induced more changes in protein secondary structure compared to melting and mixing method. SEC analysis gave evidence of protein aggregation for catalase upon adopting the wet granulation method. The biological activity of catalase was found to reduce significantly than other two proteins upon using wet granulation method, which might be ascribed to both secondary structure alterations and the formation of aggregates. Horseradish peroxidase and α-chymotrypsin did not form any soluble aggregates. In conclusion, melting and mixing method emerged as a better incorporation method compared to wet granulation because of better stability shown by the formulated proteins.

Low-pH induced structural changes, allergenicity and in vitro digestibility of lectin from black turtle bean (Phaseolus vulgaris L.)

Lectin was incubated in corresponding acidic buffers (pH 1.0–3.5) for a certain period (0.5, 1, 2, 4, 8, 12 and 24 h) at 25 °C. Low-pH induced changes in structure, allergenicity and in vitro digestibility of lectin from black turtle bean (Phaseolus vulgaris L.) were investigated in the present study. Results indicated that the alteration in structure was a progressive unfolding process mainly depending on pH environment, and the treated lectin attained a stable state at 8 h. Electrophoretic, dynamic light scattering (DLS) and size exclusion chromatography (SEC) analyses suggested that lectin monomers appeared in the solutions of pH < 2.0. Differential scanning calorimetry (DSC) confirmed that thermal stability of lectin weakened in low pH environments. Furthermore, ELISA and in vitro digestion assay showed allergenicity and digestibility significantly decreased with the structural alterations. These results showed low-pH treatments have great potential to reduce the damage of legumes protein consumption.

Characterization of thermostable alkaline protease from Bacillus halodurans SE5 and its application in degumming coupled with sericin hydrolysate production from yellow cocoon

Abstract Bacillus halodurans SE5 was newly isolated and grew well on a medium containing crude sericin extract from cocoon. Thermostable alkaline serine protease (protease_SE5) capable of decomposing sericin extract was purified to homogeneity from culture supernatant with a final yield of 25% and 2.01 × 104 U/mg. Among the six natural proteins tested, protease_SE5 showed the highest activity toward sericin. The sericin degumming, bio-bleaching coupled with sericin hydrolysate production from yellow cocoon by protease_SE5 and commercial Alcalase were demonstrated in the presence or absence of dithiothreitol (DTT). The addition of DTT enhanced the efficacy of both proteases. However, without DTT, the protease_SE5 had higher degumming ability and produced sericin hydrolysate 4-times higher than commercial enzyme based on the soluble protein concentration. SDS-PAGE and size exclusion chromatography analysis revealed that the maximal concentration of oligopeptides was observed with the hydrolysate prepared by protease_SE5 and showed higher antioxidant activity than those from Alcalase. The appreciable radical scavenging activities of the crude peptide (1.36 ± 0.07 mM) on ABTS, DPPH, and FRAP assay were 1568 ± 78, 3.6 ± 1.6, and 13.6 ± 0.4 μmol TE/L, respectively. Protease_SE5 has potential application for one step degumming and preparation of bioactive peptides from yellow cocoon.

Calcium-Induced Protein Folding in Calumenin and Calmodulin.

Binding of calcium-and small moleculesin general-often induce conformational changes in large molecules and complexes. The degree and typeof change varies, but theresulting shift in specific affinities ultimately induces a physiological response. It is therefore important for our understanding of responses at the cellular level to define coupled changes at the molecular level.Calumenin, a six-EF-hand calcium-binding protein localized in the endoplasmic reticulum, undergoes substantial calcium-induced rearrangement. We have demonstrated how calumenin changes from being unfolded in the absence of calcium to a compact trilobal fold in the presence of calcium (Mazzorana et al., PLoS One 11:e0151547, 2016).Here, we describe protocols for the expression and purification of calumenin and calmodulin, another EF-hand protein modulated by calcium, along with protocols for biophysical techniques used to characterize calcium-induced changes to protein conformation. Analytical size-exclusion chromatography in the presence and absence of calcium provides an informed indication of any larger conformational movements. Circular dichroism spectroscopy reveals alterations to the secondary or tertiary structure, while small-angle X-ray scattering explores changes further providing low-resolution conformational details.Surface plasmon resonance estimates binding kinetics and affinities completing the biophysical description of these events.

Design, synthesis, structural characterization and cell cytotoxicity of a new derivative poly(maleic anhydride-co-vinyl acetate)/miltefosine polymer/drug conjugate

In recent years, polymeric systems are selected as biomaterials because of their desired biocompatible properties and easy design/preparation of a number of different structures with lower toxicity and good solubility. Nontoxic polymeric drug carrier, maleic anhydride-co-vinyl acetate copolymer (MAVA), was prepared via free-radical chain polymerization at 80±0.1 ºC. MEK (methyl ethyl ketone) and BPO (benzoyl peroxide) were used as the organic medium and radical initiator, respectively. Copolymer was conjugated with a broad-spectrum antimicrobial agent, miltefosine (MF, an oral drug in the treatment of leishmaniosis), Impavido® and Miltex®, 1:1 molar ratio of copolymer:drug for 48 h at 60 °C in aqueous medium in presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC). Fourier transform infrared (FTIR) and nuclear magnetic resonance (1H- and 31P-NMR) were used to characterize the structure of the copolymer and MAVA/MF conjugate. Molecular weights were measured via size-exclusion chromatography (SEC). Results, obtained from the spectroscopic and SEC analysis, verified that conjugation was successfully carried out with good water-solubility. WST-1 cytotoxicity tests, 24 h by quantitative analysis, were carried out for copolymer, miltefosine, and MAVA/MF. The cytotoxicity values, by comparing with control group, were found statistically significantly different (P&lt;0.05). MAVA/MF copolymer/drug couple was successfully designed with lower cytotoxicity than the free drug (MF).

Catalyst shuttling enabled by a thermoresponsive polymeric ligand: facilitating efficient cross-couplings with continuously recyclable ppm levels of palladium.

A polymeric monophosphine ligand WePhos has been synthesized and complexed with palladium(ii) acetate [Pd(OAc)2] to generate a thermoresponsive pre-catalyst that can shuttle between water and organic phases, with the change being regulated by temperature. The structure of the polymeric ligand was confirmed with matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry and size-exclusion chromatography (SEC) analysis, as well as nuclear magnetic resonance (NMR) measurements. This polymeric metal complex enables highly efficient Pd-catalyzed cross-couplings and tandem reactions using 50 to 500 ppm palladium, and this can facilitate reactions that are tolerant to a broad spectrum of (hetero)aryl substrates and functional groups, as demonstrated with 73 examples with up to 99% isolated yields. Notably, 97% Pd remained in the aqueous phase after 10 runs of catalyst recycling experiments, as determined via inductively coupled plasma-atomic emission spectrometry (ICP-AES) measurements, indicating highly efficient catalyst transfer. Furthermore, a continuous catalyst recycling approach has been successfully developed based on flow chemistry in combination with the catalyst shuttling behavior, allowing Suzuki-Miyaura couplings to be conducted at gram-scales with as little as 10 ppm Pd loading. Given the significance of transition-metal catalyzed cross-coupling and increasing interest in sustainable chemistry, this work is an important step towards the development of a responsive catalyst, in addition to having high activity, by tuning the structures of the ligands using polymer science.

Structural and functional properties of Grb2 SH2 dimer in CD28 binding.

Growth factor receptor-bound protein 2 (Grb2) is an adaptor protein that plays a critical role in cellular signal transduction. It contains a central Src homology 2 (SH2) domain flanked by two Src homology 3 (SH3) domains. Binding of Grb2 SH2 to the cytoplasmic region of CD28, phosphorylated Tyr (pY) containing the peptide motif pY-X-N-X, is required for costimulatory signaling in T cells. In this study, we purified the dimer and monomer forms of Grb2 SH2, respectively, and analyzed their structural and functional properties. Size exclusion chromatography analysis showed that both dimer and monomer exist as stable states. Thermal stability analysis using circular dichroism showed that the dimer mostly dissociates into the monomer around 50°C. CD28 binding experiments showed that the affinity of the dimer to the phosphopeptide was about three fold higher than that of the monomer, possibly due to the avidity effect. The present crystal structure analysis of Grb2 SH2 showed two forms; one is monomer at 1.15 Å resolution, which is currently the highest resolution analysis, and another is dimer at 2.00 Å resolution. In the dimer structure, the C-terminal region, comprising residues 123–152, was extended towards the adjacent molecule, in which Trp121 was the hinge residue. The stable dimer purified using size exclusion chromatography would be due to the C-terminal helix “swapping”. In cases where a mutation caused Trp121 to be replaced by Ser in Grb2 SH2, this protein still formed dimers, but lost the ability to bind CD28.

Isolation and characterization of different promising fungi for biological waste management of polyurethanes.

SummaryAs a highly resistant polymer family, polyurethanes (PU) are responsible for increasing environmental issues. Then, PU biodegradation is a challenging way to develop sustainable waste management processes based on biological recycling. Since the metabolic diversity of fungi is a major asset for polymer degradation, nearly thirty strains were isolated from sampling on six different PU wastes‐containing environments. A screening of the fungi on four thermoplastic PU (TPU) with different macromolecular architectures led to the selection of three strains able to use two polyester PU as sole carbon source: Alternaria sp., Penicillium section Lanata‐Divaricata and Aspergillus section flavi. Weight loss, FT‐IR, Scanning Electron Microscopy and Size Exclusion Chromatography analyses revealed that these three fungi degrade slightly and similarly a fatty acid dimer‐based TPU while variability of degradation was noticed on a polycaprolactone‐based TPU. On this last TPU, robust analysis of the degraded polymers showed that the Penicillium strain was the best degrading microorganism. Membrane enzymes seemed to be involved in this degradation. It is the first time that a strain of Penicillium of the section Lanata‐Divaricata displaying PU biodegradation ability is isolated. These newly discovered fungi are promising for the development of polyester PU waste management process.

Specific oligomer recovery behavior from cured unsaturated polyester by superheated steam degradation

To examine the specific oligomer recovery behavior from cured unsaturated polyester resin (cUPR), super-heated steam (SHS) was employed as a reactant. The cUPR was treated by SHS in a temperature range of 300–350 °C and solubilized, resulting in recovery of solvent extracted residues (SE-SDRs). From size-exclusion chromatography analysis, an interesting finding was that the molecular weight (MW) value of SE-SDR, increased gradually with degradation time. This specific recovery behavior was analyzed by FT-IR and 1H NMR spectroscopies. Based on the analytical results, a proposed SHS degradation mechanism was that first orthophthalate and glycol ester groups were degraded and removed as short length fragments, after which cleavage of styrene unit crosslinkers induced collapse of the 3-D network structure with the resulting solubilizing cUPR as SE-SDR. This mechanism can explain the specific oligomer recovery behavior whereby an increase in MW of SE-SDR occurred with SHS-degradation time. Obtained styrene-fumarate copolymer having Mn 2550 and Mw 9480 was recovered in a maximum 57 wt% yield after SHS treatment at 325 °C for 180 min.

Analysis of Network Structures in Thiol-Ene UV Curing System Using Reworkable Resins.

An analysis of the network structures in thiol-ene UV curing resins was carried out using reworkable resins composed of di(meth)acrylate monomers having tertiary ester linkages. The effect of the functionality of the thiols, the functional ratio of the thiol and ene functions, their conversions and curing atmosphere on the chain lengths was discussed. A mixture of (meth)acrylates, thiol compounds, a photoradical initiator, and a photoacid generator was cured by irradiation at 365 nm. The cured samples were degraded by heating after irradiation at 254 nm. Size exclusion chromatography (SEC) and 1H NMR analyses of the degraded samples were carried out after the methylation. The crosslinking conditions strongly affected the network structures. The degraded samples have molecular weights between 250 and 2700. The molecular weights of the degraded resins increased with the functionality of the thiol compounds. The chain length dependence suggests that thiol compounds with a high functionality have a low reactivity due to steric hindrance. The chain lengths of the degraded networks were nearly proportional to the concentration of the (meth)acrylate monomers. The addition of reactive diluents enhanced the reactivity and increased the chain length.

Reduction of chromatin heteroaggregates by acid precipitation of mammalian cell culture and ramification in protein A chromatography for recombinant immunoglobulin G purification

Acid precipitation has been demonstrated as a clarification method for cell culture supernatant (CCS) pretreatment during Immunoglobulin G (IgG) purification. In this study, the responses of IgG, non-histone host cell protein (n-hHCP), histone, and DNA to different pHs in the presence of salt (NaCl) at various concentrations were systematically studied. IgG loss was closely associated with the precipitation of chromatin heteroaggregates, in which DNA/histone showed significant decreases along with the acidification of buffer environment. In addition, DNA showed full recovery after pH neutralization, while the low pH treatment induced histone precipitation was permanent and could not be reverted upon pH increase. Analytical size exclusion chromatography (SEC) profiles further indicated the agglomeration and reduction of chromatin heteroaggregates along with lower pH. Protein A chromatography benefited from the reduction of chromatin heteroaggregates and showed significant enhancement of the removal of impurities as well as for IgG recovery. For the first time, this study correlated CCS acidification with chromatin heteroaggregate removal and illustrated its ramification for the subsequent purification process.

Fine-fibrous cellulose II aerogels of high specific surface from pulp solutions in TBAF·H2O/DMSO

Abstract Lightweight cellulose II aerogels featuring densities of about 40–70 mg cm−3 were prepared from 1 to 3% solutions of different pulps in hot (60°C) tetra-n-butylammonium fluoride (TBAF)·H2O/dimethyl sulfoxide (DMSO) by (i) the coagulation of cellulose with EtOH to afford self-standing, transparent and homogeneous gels, (ii) gel ripening and washing, (iii) solvent exchange and (iv) supercritical carbon dioxide (scCO2) drying. Size exclusion chromatography (SEC) analyses confirmed that the chemical integrity of cellulose is largely preserved at short dissolution times. Dissolution of more than 2% of cellulose at a sufficiently low viscosity for solution, casting was achieved after the water content of TBAF was reduced to a value equaling that of the monohydrate. Intriguingly, the obtained aerogels featured higher specific surfaces (≤470 m2 g−1) than comparable materials prepared from other cellulose solvents. This is due to the particular morphology of TBAF aerogels, which is supposedly formed by spinodal decomposition of the cellulose/solvent mixture upon exposure to the cellulose antisolvent. As a result, largely homogeneous three-dimensional (3D) networks of agglomerated cellulose spheres were formed, which simultaneously acted as supporting scaffolds for interconnected micron-size voids. As cellulose spheres are composed of very small interwoven nanofibers, TBAF-derived aerogels contain a high portion of micropores and small amounts of mesopores, too.

Synthesis of biobased polyols using algae oil for multifunctional polyurethane coatings

Renewable sources – namely, algae oil and diacids – were used for the synthesis of alkyd (ALK) and polyesteramide (PEA) polyols. The prepared polyols were characterised by Fourier transform infrared and nuclear magnetic resonance techniques and size exclusion chromatography analysis. After reaction with diisocyanate, polyols were converted to polyurethane (PU) coatings, which were then characterised by X-ray diffraction, differential scanning calorimetry and thermogravimetric analysis. The biocidal activities of the films were evaluated against Gram-negative (Escherichia coli ) and Gram-positive bacteria (Staphylococcus aureus) by using the turbidity method as well as by observation of film degradation or biofilm formation by bacteria on the surface of a metal tray by using scanning electron microscopy. The PUs obtained using ALK and PEA showed better antimicrobial properties compared with PUs based on algae oil fatty amide (AOFA), monoglyceride (MG) and even an uncoated mild steel (MS) panel. The anticorrosion properties of PU coatings were evaluated by the potential dynamic polarisation (PDP) method in a 3·5 wt% sodium chloride (NaCl) corrosive media. All coated samples (ALK and PEA) showed good barrier properties against corrosion compared with AOFA, MG and the uncoated MS panel. The prepared algae-oil-based PU coatings showed better chemical, mechanical, water-repellent, antimicrobial, anticorrosive and thermal properties.

One-step affinity purification of fusion proteins with optimal monodispersity and biological activity: application to aggregation-prone HPV E6 proteins

BackgroundBacterial expression and purification of recombinant proteins under homogeneous active form is often challenging. Fusion to highly soluble carrier proteins such as Maltose Binding Protein (MBP) often improves their folding and solubility, but self-association may still occur. For instance, HPV E6 oncoproteins, when produced as MBP-E6 fusions, are expressed as mixtures of biologically inactive oligomers and active monomers. While a protocol was previously developed to isolate MBP-E6 monomers for structural studies, it allows the purification of only one MBP-E6 construct at the time. Here, we explored a parallelizable strategy more adapted for biophysical assays aiming at comparing different E6 proteins.ResultsIn this study, we took advantage of the distinct size and diffusion properties of MBP-E6 monomers and oligomers to separate these two species using a rapid batch preparation protocol on affinity resins. We optimized resin reticulation, contact time and elution method in order to maximize the proportion of monomeric MBP-E6 in the final sample. Analytical size-exclusion chromatography was used to quantify the different protein species after purification. Thus, we developed a rapid, single-step protocol for the parallel purification of highly monomeric MBP-E6 samples. MBP-fused HPV16 E6 samples obtained by this approach were validated by testing the binding to their prototypical peptide targets (the LXXLL motif from ubiquitine ligase E6AP) by BIAcore-SPR assay.ConclusionsWe have designed a rapid single-step batch affinity purification approach to isolate biologically active monomers of MBP-fused E6 proteins. This protocol should be generalizable to isolate the monomer (or the minimal biologically active oligomer) of other proteins prone to self-association.

Proteolytic regulation of the extent of dietary proteins with skin grape proanthocyanidin and anthocyanidin's interactions

SummaryDuring technological processing, proanthocyanidins and anthocyanidins could be partly lost due to the complexation phenomena, affecting food and beverage nutritional properties, organoleptic properties and health‐promoting potentials. A common issue is encountered when processing food and beverage which is binding of phenolics to dietary proteins. The present investigation aims at evaluating the proteolysis contribution, using pure protease (Pepsin, 3000 units g−1), to protein–anthocyanidin and protein–proanthocyanidin interactions. Bovine serum albumin (BSA), ovalbumin (OVA) and casein (CAS) dietary protein models were used. High‐performance liquid chromatography coupled to diode array detector (HPLC‐DAD) and size exclusion chromatography analyses proved that pepsin treatment significantly (P &gt; 0.05) decreased the ratio of flavonoids’ interaction with tested proteins . The proteolysis reduced anthocyanidin interactions with CAS, OVA and BSA by 64.88%, 57.37% and 42.87% respectively. Similarly, proanthocyanidins interaction with CAS, OVA and BSA were reduced by 34.23%, 13.74% and 2.39% respectively. This study provides the basis to develop innovative technologies to limit protein–flavonoid complexation during food and beverage processing.