Human Transferrin Research Articles

Production of Recombinant Human Transferrin in Eukaryotic Pichia pastoris Expression System.

The development and manufacturing of serum-free culture media allowing reducing the costs of preparations and standardizing the biotechnological process are important trends in biotechnology. Substitution of protein compounds in the serum-free media with recombinant analogues reduces the risk of contamination with various infectious agents. Human transferrin is a protein component of serum-free media responsible for the transport of Fe3+ ions into cells. We generated a producing strain P. pastoris secreting human transferrin to the culture medium. The use of constitutive GAP promoter and maintenance of medium pH at 6.5 allows attaining maximum level of transferrin expression (20 mg/liter).

The heme-sensitive regulator SbnI has a bifunctional role in staphyloferrin B production by Staphylococcus aureus

Staphylococcus aureus infection relies on iron acquisition from its host. S. aureus takes up iron through heme uptake by the iron-responsive surface determinant (Isd) system and by the production of iron-scavenging siderophores. Staphyloferrin B (SB) is a siderophore produced by the 9-gene sbn gene cluster for SB biosynthesis and efflux. Recently, the ninth gene product, SbnI, was determined to be a free l-serine kinase that produces O-phospho-l-serine (OPS), a substrate for SB biosynthesis. Previous studies have also characterized SbnI as a DNA-binding regulatory protein that senses heme to control sbn gene expression for SB synthesis. Here, we present crystal structures at 1.9-2.1 Å resolution of a SbnI homolog from Staphylococcus pseudintermedius (SpSbnI) in both apo form and in complex with ADP, a product of the kinase reaction; the latter confirmed the active-site location. The structures revealed that SpSbnI forms a dimer through C-terminal domain swapping and a dimer of dimers through intermolecular disulfide formation. Heme binding had only a modest effect on SbnI enzymatic activity, suggesting that its two functions are independent and structurally distinct. We identified a heme-binding site and observed catalytic heme transfer between a heme-degrading protein of the Isd system, IsdI, and SbnI. These findings support the notion that SbnI has a bifunctional role contributing precursor OPS to SB synthesis and directly sensing heme to control expression of the sbn locus. We propose that heme transfer from IsdI to SbnI enables S. aureus to control iron source preference according to the sources available in the environment.

Effect of Pegylation and Targeting Moieties on the Ultrasound-Mediated Drug Release from Liposomes.

The use of targeted liposomes encapsulating chemotherapy drugs enhances the specific targeting of cancer cells, thus reducing the side effects of these drugs and providing patient-friendly chemotherapy treatment. Targeted pegylated (stealth) liposomes have the ability to safely deliver their loaded drugs to the cancer cells by targeting specific receptors overly expressed on the surface of these cells. Applying ultrasound as an external stimulus will safely trigger drug release from these liposomes in a controlled manner. In this study, we investigated the release kinetics of the model drug "calcein" from targeted liposomes sonicated with low-frequency ultrasound (20 kHz). Our results showed that pegylated liposomes were more sonosensitive compared to nonpegylated liposomes. A comparison of the effect of three targeting moieties conjugated to the surface of pegylated liposomes, namely human serum albumin (HSA), transferrin (Tf) and arginylglycylaspartic acid (RGD), on calcein release kinetics was conducted. The fluorescent results showed that HSA-PEG and Tf-PEG liposomes were more sonosensitive (showing higher calcein release following the exposure to pulsed LFUS) compared to the control pegylated liposomes, thus adding more acoustic benefits to their targeting efficacy.

Isolation of proliferating cells from whole blood using Human Transferrin Receptor in a two-stage separation system

Blood is a routinely tested biological fluid for diagnosis and monitoring of diseases as many diseases would trigger a change in white blood cell count. Thus, several methods have been established to isolate or enrich white blood cells from patient blood samples for such analyses. One method of preparing an enriched white blood cell sample is through the selective lysis of red blood cells by hypotonic shock and restoration of osmolarity to maintain viability of target white blood cells. An inherent problem with this approach is the loss of target cells during sample handling. We report a two-stage separation system that can perform lysis and restoration of osmolarity of blood on-chip and direct the resultant sample to the second step of the analysis. Hence, there is no loss of sample. The post-lysis makeup features a protein-rich buffer to help stabilize cells. As proof of concept, we spiked HL-60 cells into a whole blood and a pre-lysed blood sample and compared capture metrics of each method using a downstream affinity separation. The capture efficiency of the whole blood sample ranged between 40 and 80% using <7 μL of sample compared to 10–52% from 60 μL of pre-lysed blood required for similar analysis. In addition, both pre-lysed and whole blood samples showed no significant difference in purity and viability. This two-stage separation system has demonstrated the capacity to replace centrifugation and wash steps required for the preparation of lysed blood, for white blood cell analyses.

A New titania glyco-purification tip for the fast enrichment and efficient analysis of glycopeptides and glycans by MALDI-TOF-MS

Enrichment and/or purification of intact glycopeptides and derivatized glycans are essential for mass spectrometry-based glycoproteomics and glycomics because of analytical challenges observed in the analysis. Here, a titania-based material was synthesized using a facile sol-gel method for the efficient analysis of glycopeptides and glycans. The glycopeptide enrichment efficacy of the titania-based sol-gel material was evaluated by the enrichment of IgG N-glycopeptides from their proteolytic products. The selectivity and sensitivity (0.5 fmol μL−1) of the titania-based sol-gel material was found to be quite high when compared with commercial TiO2. A pipette-tip application for rapid glycoproteomic and glycomic analysis was developed by packing titania-based material into the pipette tips. The released and derivatized N-glycans of human plasma, IgG and human transferrin were successfully purified by titania glyco-purification tips for the analysis by MALDI-MS. The enrichment performance of the titania glyco-purification tip was tested using human plasma digest as a real-world complex sample and, 112 N-glycopeptides from human plasma glycoproteins were detected by the analysis of enriched samples by nLC-QTOF-MS/MS. Finally, the structural characterization of the material was achieved in detail using various characterization approaches.

IgA1 isolated from Henoch-Schönlein purpura children promotes proliferation of human mesangial cells in vitro.

Previous studies show that the proliferation of human mesangial cells (HMCs) played a significant part in the pathogenesis of Henoch-Schönlein purpura nephritis (HSPN). The aim of this study was to explore the proliferation of HMCs induced by IgA1 isolated from the sera of HSP patients. HMCs were cultured in three different types of media, including IgA1 from patients with HSP (HSP IgA1 group), healthy children (healthy IgA1 group) and medium (control group). The proliferation of HMCs incubated with IgA1 was determined by cell counting kit-8 assay and bromodeoxyuridine incorporation. The expression of ERK1/2 and phosphatidylinositol 3 kinase/protein kinase B/mammalian targets of the rapamycin (PI3K/AKt/mTOR) signals and transferrin receptor (TfR/CD71) was detected with the methods of immunoblotting. The results indicated that the proliferation of HMCs significantly increased in the HSP IgA1 group compared with that in the control group or the healthy IgA1 group (P < 0.001). Moreover, we found that IgA1 isolated from HSP patients activated ERK and PI3K/AKt/mTOR signals, and markedly increased TfR/CD71 expression in HMCs. These effects induced by IgA1 isolated from patients with HSP were inhibited by human TfR polyclonal antibody (hTfR pAb) and soluble human transferrin receptor (sTfR), indicating that IgA1-induced HMC proliferationand ERK1/2 and PI3K/AKt/mTOR activation were dependent on TfR/CD71 engagement. Altogether, these data suggested that TfR/CD71 overexpression and ERK1/2 and PI3K/AKt/mTOR activation were engaged in HMC proliferation induced by IgA1 from HSP patients, which might be related to the mesangial injury of HSPN.

The effect of protein expression on cancer cell capture using the Human Transferrin Receptor (CD71) as an affinity ligand

Cancer cell detection in liquid biopsies has been a widely studied application in many microfluidic devices. The use of a common antibody, such as the epithelial cell adhesion molecule (Anti-EpCAM) or other specific antibodies, has facilitated the detection and study of many cancers. However, the use of such antibodies requires a priori knowledge of the cancer source, and many cancer subtypes are missed in screening applications. There remains a need to study a wider range of cancers that maintain the streamlined antibody approach in cell affinity separations. The Human transferrin receptor (CD71) has recently been demonstrated as a cancer cell affinity target in blood samples. CD71 expression in blood cells is low, whereas proliferating cancer cells have a higher expression of the surface protein. CD71 expression is variable with cell cycle, which can impact cell separations. In this work, we investigated the effects of cell cycle and CD71 expression on cell capture metrics. Six cancer cell lines were isolated from blood via CD71 affinity capture, with a capture efficiency and purity that varied with CD71 expression. Despite variation in CD71 expression, the affinity was sufficient to isolate cancer cells spiked into blood; under optimal conditions, CD71-based capture resulted in capture purity >80%. We conclude that CD71 affinity separations show potential as a biomarker for cancer studies without sacrificing sensitivity and selectivity, and that cancer cells can be isolated from liquid biopsies over a range of expression of the target protein.

Diagnostic Potential of Transferrin Glycoforms-A Lectin-Based Protein Microarray Approach.

Disease or a specific condition may cause alteration of human transferrin (hTf) glycosylation pattern. A specific analytical platform, lectin-based protein microarray, is designed and optimized for the investigation of hTf glycans, attached to the protein core in their native form. hTf molecules isolated from healthy persons of different age, diabetes mellitus type 2 (T2DM) or colorectal carcinoma (CRC) patients are used for method validation. Reliability of the results is ensured by three criteria for the evaluation of hTf-lectin interactions: i) signal-to-noise ratio above 3, ii) signal intensity above 250 arbitrary units, and iii) hTf concentration ensuring high sensitivity of the assay. Six lectins, out of fourteen tested, satisfy the criteria. hTf is spotted at concentration of 50µg mL-L . When physiological samples (isolated hTf) are analyzed, the highest potential to differentiate between population groups expresses Aleuria aurantia (AAL), Triticum vulgaris (WGA) and Phaseolus vulgaris (PHA-E) lectins. The initial amount of hTf which can be analyzed is very low (75 pg). Results confirm that a very sensitive, high-throughput lectin-based protein microarray platform can be formulated to detect changes in hTf glycan structures which can be considered as biomarkers of ageing or a disease.

Formation of a Monolayer Protein Corona around Polystyrene Nanoparticles and Implications for Nanoparticle Agglomeration.

Nanoparticle (NP) interactions with cells and organisms are mediated by a biomolecular adsorption layer, the so-called "protein corona." An in-depth understanding of the corona is a prerequisite to successful and safe application of NPs in biology and medicine. In this work, earlier in situ investigations on small NPs are extended to large polystyrene (PS) NPs of up to 100 nm diameter, using human transferrin (Tf) and human serum albumin (HSA) as model proteins. Direct NP sizing experiments reveal a reversibly bound monolayer protein shell (under saturating conditions) on hydrophilic, carboxyl-functionalized (PS-COOH) NPs, as was earlier observed for much smaller NPs. In contrast, protein binding on hydrophobic, sulfated (PS-OSO3 H) NPs in solvent of low ionic strength is completely irreversible; nevertheless, the thickness of the observed protein corona again corresponds to a protein monolayer. Under conditions of reduced charge repulsion (higher ionic strength), the NPs are colloidally unstable and form large clusters below a certain protein-NP stoichiometric ratio, indicating that the adsorbed proteins induce NP agglomeration. This comprehensive characterization of the persistent protein corona on PS-OSO3 H NPs by nanoparticle sizing and quantitative fluorescence microscopy/nanoscopy reveals mechanistic aspects of molecular interactions occurring during exposure of NPs to biofluids.

Development of a filter-aided extraction method coupled with glycosylamine labeling to simplify and enhance high performance liquid chromatography-based N-glycan analysis

Efficient sample pretreatment of N-glycans from glycoproteins is essential but challenging due to the limitations of existing tedious and laborious methods in N-glycomics. This study aimed to establish a filter-aided extraction method coupled with glycosylamine AQC labeling for a simple and rapid direct HPLC-FLD-based analysis of N-glycans. The developed method was demonstrated to be simpler and more sensitive compared to previous HILIC SPE purification method coupled with glycosylamine labeling. It has been validated with wild-type N-glycans from human transferrin and RNase B and then was successfully applied to investigate N-glycan profiles of the transferrin in human serum and a monoclonal antibody (mAb). Results showed good applicability of the method for complex samples. Additionally, this method is compatible with the replicate determination of N-glycan samples to assess the high-throughput analysis of glycan variability in mAb sample.

Indinavir-Loaded Nanostructured Lipid Carriers to Brain Drug Delivery: Optimization, Characterization and Neuropharmacokinetic Evaluation.

As an anti-retroviral Protease Inhibitor (PI), Indinavir (IDV) is part of the regimen known as Highly Active Anti-Retroviral Therapy (HAART) widely used for Human Immunodeficiency Virus (HIV) infection. The drug efficiency in treatment of the brain manifestations of HIV is, however, limited which is mainly due to the efflux by P-glycoprotein (P-gp) expressed at the Blood-Brain Barrier (BBB). To overcome the BBB obstacle, NLCs were used in this study as carriers for IDV, which were optimized through two steps: a "one-factor-at-a-time" screening followed by a systematic multiobjective optimization. Spherical smooth-surfaced Nanoparticles (NPs), average particle size of 161.02±4.8 nm, Poly-Dispersity Index (PDI) of 0.293±0.07, zeta potential of -40.62±2.21 mV, entrapment efficiency of 93±1.58%, and loading capacity of 9.15±0.15% were obtained after optimization which were, collectively, appropriate in terms of the objective of this study. The surface of the optimized NPs was, then, modified with human Transferrin (TR) to improve the drug delivery. The particle size, zeta potential, and PDI of the TR-modified NLCs were 185.29±6.7nm, -28.68±3.37 mV, and 0.247±0.06, respectively. The in vitro release of IDV molecules from the NPs was best fitted to the Weibull model indicating hybrid diffusion/erosion behavior. As the major in vivo findings, compared to the free drug, the NLCs and TR-NLCs displayed significantly higher and augmented concentrations in the brain. In this case, NLC and TR-NLC were 6.5- and 32.75-fold in their values of the brain uptake clearance compared to free drug.

Use of 19 F Differential Labelling for the Simultaneous Detection and Monitoring of Three Individual Proteins in a Serum Environment.

Protein behavior in complex mixtures, such as biological fluids, is often modeled by simplified buffer systems in solution. Here we have used the recently described differential 19 F labelling approach (with NMR detection) to monitor and compare the solution behaviour of three proteins at once: human serum albumin (HSA), transferrin (TrF), and immunoglobulin G (IgG), both in serum and in buffer. We demonstrate that monitoring three proteins simultaneously and independently in biological fluid is possible, and that the presence of other endogenous components greatly changes the association characteristics of these proteins. For example, in the simplified model buffer system, all three proteins diffuse at a similar rate, while in serum HSA diffuses around three times faster than TrF, and four times faster than IgG. This 19 F NMR approach allows characterization of the behaviour of complex multiprotein systems in their native environment, e. g., in biological fluids.

Spectroscopic, calorimetric and molecular docking insight into the interaction of Alzheimer’s drug donepezil with human transferrin: implications of Alzheimer’s drug

Donepezil, a highly selective and reversible inhibitor, is amongst the widely used drugs for the treatment of Alzheimer’s disease (AD) across the globe. Clinically significant human transferrin (hTf) is a key player involved in iron metabolism. The current study targets to have an insight into the interaction between donepezil and this plasma protein, hTf using UV–vis absorbance, fluorescence, isothermal titration calorimetry, circular dichroism (CD) and molecular docking techniques. UV spectroscopy of hTf in the presence of donepezil revealed the occurrence of hyperchromism coupled with blue shift in indicating complex formation. Binding constant obtained from UV–vis absorption measurements was of the order 0.9 × 104 M−1 implicative of strong binding. Fluorescence spectroscopy shows that donepezil quenches fluorescence intensity of the native hTf in dynamic manner with a shift in wavelength maxima. Donepezil binds to hTf at 310 K with a binding constant of 1.3 × 104 M−1 suggestive of strong binding. ITC results clearly suggest the reaction to be spontaneous and thermodynamically favorable. CD spectroscopy also showed a shift in the spectrum of the native hTf in the presence of donepezil suggesting formation of complex between protein and drug. Furthermore, molecular docking experiments showed Pro 91, Phe 94 and His 207 to be key players involved in the complex formation. This study gives an insight into the molecular basis of interaction between donepezil and hTf thereby aiding in understanding the activity and mechanism of protein and drug binding. This will serve as a boon for drug designing industries which are working day and night for improved treatment of existing disorders across the world.Communicated by Ramaswamy H. Sarma

Competition between Al3+ and Fe3+ binding to human transferrin and toxicological implications: structural investigations using ultra-high resolution ESI MS and CD spectroscopy.

Human serum transferrin (hTF) is an iron binding protein with the primary task of ensuring well-controlled transport of Fe3+-ions in the bloodstream. Furthermore, hTF has been identified as a key component in the trafficking of Al3+-ions from the serum to cells. It is clear that binding alone does not guarantee cellular uptake via the transferrin receptor, since this is determined by the structural properties of the metal-protein complex. The conformation of the metallated hTF is critically important for delivery of Fe3+ or any other metal into the cell. The combination of ultra-high resolution ESI mass spectrometry and CD spectroscopy together provide accurate species distribution of the Fe3+ during stepwise addition to apo-hTF and an indirect indication of the tertiary structure of the metallated protein. These two methods together are extremely fine probes of structural changes as a function of precise metal binding status at micromolar concentrations. Simulation of the precise domain distribution could be determined during the stepwise metallation from 0 to 2 Fe3+ added. Analysis of the ESI-MS data for the stepwise metallation of apo-hTF and Al1 or 2-hTF with Fe3+ was carried out and used to simulate the experimental speciation based on the reported KF values. There are six main conclusions: (1) Fe3+ binds predominantly, initially to the C-lobe. (2) The CD spectral properties indicate that the C-lobe metallation dominates the structural properties of both binding sites; N-lobe metallation modifies the C-lobe structure. (3) Fe3+ metallation of the mixed Al1-2-hTF results in the dominant form of Fe1Al1-hTF. (4) The first Fe3+ bound to Al1-hTF binds predominantly in the C-lobe domain. (5) The CD spectral properties when Fe3+ binds to Al1-2-hTF indicates that Al-N-lobe occupation mirrors the structural effects of N-lobe occupation by Fe3+. (6) With respect to how Al3+ might enter the cell, the formation of a hybrid form Al1Fe1-hTF might enable the Al3+ to enter the cell via receptor-mediated endocytosis due to the binding of Fe3+ in the C-lobe of the protein which is primarily responsible for the structure of the metal-protein complex.

Asialo-transferrin: Biochemical aspects and association with alcohol abuse investigation.

Asialo-human transferrin (asialo-hTf) is a glycoform of the human serum protein transferrin characterized by the lack of the sialic acid (SA) terminal unit. It is known that glycosylation micro-heterogeneity and the presence of SA are strongly involved in protein functioning and pathophysiological activities. Some hTf glycoforms are valuable biomarkers for the detection of both genetic defects of glycosylation and/or sialoform distribution changes. The detection of the carbohydrate deficient transferrin (CDT) glycoforms is currently a widely employed method for the diagnosis of chronic alcohol abuse. The physiological significance of asialo-hTf is still unclear, despite its important biological implications. The current knowledge suggests that asialo-hTf may be involved in regulation of iron transport and release at the hepatic level, which, consequently, could strongly be affected by alcohol consumption. For these reasons, a deeper understanding of asialo-hTf structure and its physiological role is required, and an improved method of its analysis would favor the detection of both chronic abuse and other habits of alcohol intake and/or misuse. Thus, suitable analytical methods possessing higher sensitivity and specificity in comparison with the currently available techniques are certainly recommended. The present review summarizes the studies on asialo-hTf structure, roles, and detection techniques mainly in relation to its possible use as a potentially additional useful biomarker of alcohol abuse, and underlines its prospective value as a forensic and diagnostic tool.

Binding of Cm(III) and Th(IV) with Human Transferrin at Serum pH: Combined QM and MD Investigations.

Human serum transferrin (sTf) can also function as a noniron metal transporter since only 30% of it is typically saturated with a ferric ion. While this function of sTf can be fruitfully utilized for targeted delivery of certain metal therapeutics, it also runs the risk of trafficking the lethal radionuclides into cells. A large number of actinide (An) ions are known to bind to the iron sites of sTf although molecular-level understanding of their binding is unclear. Understanding the radionuclide interaction with sTf is a primary step toward future design of their decorporating agents since irrespective of the means of contamination, the radionuclides are absorbed and transported by blood before depositing into target organs. Here, we report an extensive multiscale modeling approach of two An (curium(III) and thorium(IV)) ions' binding with sTf at serum physiological pH. We find that sTf binds both the heavy ions in a closed conformation with carbonate as synergistic anions and the An-loaded sTf maintains its closed conformation even after 100 ns of equilibrium molecular dynamics (MD) simulations. MD simulations are performed in a polarizable water environment, which also incorporates electronic continuum corrections for ions via charge rescaling. The molecular details of the An coordination and An exchange free energies with iron in the interdomain cleft of the protein are evaluated through a combination of quantum mechanical (QM) and MD studies. In line with reported experimental observations, well-tempered metadynamics results of the ions' binding energetics show that An-sTf complexes are less stable than Fe-sTf. Additionally, curium(III) is found to bind more weakly than thorium(IV). The latter result might suggest relative attenuation of thorium(IV) cytotoxicity when compared with curium(III).

Cryo-EM structure of the human ferritin\u2013transferrin receptor 1 complex

Human transferrin receptor 1 (CD71) guarantees iron supply by endocytosis upon binding of iron-loaded transferrin and ferritin. Arenaviruses and the malaria parasite exploit CD71 for cell invasion and epitopes on CD71 for interaction with transferrin and pathogenic hosts were identified. Here, we provide the molecular basis of the CD71 ectodomain-human ferritin interaction by determining the 3.9 Å resolution single-particle cryo-electron microscopy structure of their complex and by validating our structural findings in a cellular context. The contact surfaces between the heavy-chain ferritin and CD71 largely overlap with arenaviruses and Plasmodium vivax binding regions in the apical part of the receptor ectodomain. Our data account for transferrin-independent binding of ferritin to CD71 and suggest that select pathogens may have adapted to enter cells by mimicking the ferritin access gate.

Molecular-Level Profiling of Human Serum Transferrin Protein through Assessment of Nanopore-Based Electrical and Chemical Responsiveness.

In this study, we investigated the voltage and pH responsiveness of human serum transferrin (hSTf) protein using silicon nitride (Si xN y) nanopores. The Fe(III)-rich holo form of hSTf was dominant when pH > pI, while the Fe(III)-free apo form was dominant when pH < pI. The translocations of hSTf were purely in an electrophoretic sense, thus depended on its pI and the solution pH. With increasing voltage, voltage driven protein unfolding became prominent which was indicated by the trends associated with change in conductance, due to hSTf translocation, and in the excluded electrolyte volume. Additionally, analysis of the translocation events of the pure apo form of hSTf showed a clear difference in the event population compared to that of the holo form. The results obtained demonstrate the successful application of nanopore devices to distinguish between the holo and apo forms of hSTf in a mixture and to analyze its folding and unfolding phenomenon over a range of pH and applied voltages.

Visualized detection of apo-transferrin based on cyanine dye supramolecular assembly.

In this work, a new biological probe for human transferrin (hTf) detection based on a cyanine dye ETC supramolecular assembly was designed. In sub-micromolar level, apo-hTf could induced the ETC aggregations transferred from H-aggregations to J-band with color change from pink to blue, while holo-hTf hardly possessed the ability, indicating ETC could specifically identify apo-hTf. The present study allowed for apo-hTf detection in the range of 8-80 nM with a detection limit of 2.8 nM and the sensitivity of visualization was around 70 nM. To further examine the suitability, iron ions were added into apo-hTf to stimulate the transformation from open conformation to the closed one gradually. It has been confirmed through tryptophan internal fluorescence quenching and the decrease of ETC J-aggregation. The interaction between ETC and apo-hTf performed high affinity that Ka was reached to 106 M-1 with a high selectivity. The potential in practical applications of this method has been tested for detection apo-hTf in human serum.

On/off resonance Raman optical activity of human serum transferrin

AbstractThe transferrin proteins exhibit two high affinity Fe(III) binding pockets and are responsible for the iron transport into cells of higher order organisms. Previously, transferrins have been studied as possible transporter molecules for drugs delivery. In this study, Raman optical activity (ROA) was employed to investigate human serum transferrin. Due to the presence of Fe(III) in the holo form of the protein, it is in resonance with the the laser excitation wavelength (of 532 nm) used in the experiments. Consequently, resonance enhanced ROA bands were observed in the spectrum of the holo form. Nevertheless, far from resonance bands, arising from the protein backbone, could simultaneously be observed in the ROA spectrum of the holo form. This implies that ROA can be used to provide simultaneously information on the metal binding pocket as well as on the secondary structure of the protein. Furthermore, it was found that the amount of observed resonance enhanced ROA signals can be tuned by partially removing the iron present in the protein. Electronic circular dichroism (ECD) was employed to verify the Raman/ROA results.