Tracking Heme-Protein Interactions in Healthy and Pathological Human Serum in Native Conditions by Miniaturized FFF-Multidetection

Abstract

The interaction of heme with blood serum proteins plays an important role in many physiological and pathological processes involving enzyme activity, gene expression and cell proliferation. The mechanisms underlying these interactions are; however, not yet fully understood. New analytical methods able to investigate protein-heme binding in native, biologically representative conditions are thus required. In this work, we present a method based on miniaturized, hollow-fiber flow field-flow fractionation with multiple spectrophotometric and light-scattering detection for size separation of high-abundance serum proteins and selective detection of heme-bound subpopulations. Heme is found to mainly interact with serum albumin, whereas a low amount also binds to other proteins such as IgM. The ability to bind heme in physiological conditions is also investigated for individual serum proteins. IgG is found unable to bind heme at clinically relevant concentrations. The proposed method allows separation, quantitation, and mass/size characterization of serum high-abundance proteins, providing information of heme-protein complex stability and preferred heme-clearing pathways. The same approach could be in perspective extended to the investigation of specific heme-antibody binding, and to further studies involving other molecules of pharmaceutical/clinical interest.

Preparation of Temperature-Responsive Antibody–Nanoparticles by RAFT-Mediated Grafting from Polymerization

“…To confirm the polymerization of NIPAAm from IgG in more detail, the prepared samples were measured using FFF. The FFF technique is a class of analytical techniques that separate analytes based on their hydrodynamic radius owing to the combined action of the laminar flow of a carrier solution, named elution flow, and the flow applied orthogonally to the elution flow, known as cross flow [ 34 ]. Figure 3 shows the FFF profiles obtained by measuring the UV absorbance of the samples at 280 nm before and after the conjugation of IgG with PNIPAAm.…”

A Green Analytical Method Combined with Chemometrics for Traceability of Tomato Sauce Based on Colloidal and Volatile Fingerprinting

“…Analytes with lower diffusivity (thus higher r h ) tend to accumulate near the accumulation wall, while smaller nano systems diffuse towards higher laminar flows. In addition to the separative AF4 analysis, we conducted two other non-separative experiments: Flow Injection Analysis (FIA), and Focus-FIA (FFIA) [ 41 , 42 ]. An FIA is a shorter, non-separative, non-filtering analysis where the signal is related to the whole sample content.…”

“…Carrier fluids, pH, and salinity can be adjusted to match the required environment, while the separation device geometry allows for simultaneous filtration of ions and small molecules thus leading to the selective characterization of the colloidal particles [ 34 , 35 , 37 , 38 ]. Moreover, AF4 multi-detection platforms can include various detectors such as UV-Vis, fluorescence, and multi-angle light scattering (MALS) to provide sample composition and spectroscopic properties, monitor stability, and investigate aggregation and conjugate formation [ 39 , 40 , 41 ]. To date, AF4 has been employed on numerous samples such as biocompatible nanoparticles [ 42 , 43 ], plant-derived proteins, and biological samples in the native state [ 44 , 45 , 46 , 47 ].…”

Extracting Information and Enhancing the Quality of Separation Data: A Review on Chemometrics-Assisted Analysis of Volatile, Soluble and Colloidal Samples

“…FFF technologies are characterized by an higher operational range than SEC, about 15 orders of magnitude mass-wise corresponding to a few nanometers to about 100 µ m, and possess a series of characteristics unmatched by other separation techniques such as: (1) Extreme versatility both in terms of mobile phase and sample injectable volume, allowing it to work in conditions close to the native ones; (2) absence of stationary phase preventing undesired interactions between the samples components of the separative system. These features allowed FFF techniques to be exploited in a wide range of applications from the separation and characterization (mass, size and spectroscopic properties) of drug carriers [165,166], antimicrobial agents [167,168], biological samples of varying complexity [169][170][171] and other products of pharmaceutical interest [5,160].…”