Purification Of Hemoglobin Research Articles

Cu(II)-Loaded Polydopamine-Coated Urchin-like Titanate Microspheres as a High-Performance IMAC Adsorbent for Hemoglobin Separation.

Immobilized metal ion affinity chromatography (IMAC) adsorbents generally have excellent affinity for histidine-rich proteins. However, the leaching of metal ions from the adsorbent usually affects its adsorption performance, which greatly affects the reusable performance of the adsorbent, resulting in many limitations in practical applications. Herein, a novel IMAC adsorbent, i.e., Cu(II)-loaded polydopamine-coated urchin-like titanate microspheres (Cu-PDA-UTMS), was prepared via metal coordination to make Cu ions uniformly decorate polydopamine-coated titanate microspheres. The as-synthesized microspheres exhibit an urchin-like structure, providing more binding sites for hemoglobin. Cu-PDA-UTMS exhibit favorable selectivity for hemoglobin adsorption and have a desirable adsorption capacity towards hemoglobin up to 2704.6 mg g-1. Using 0.1% CTAB as eluent, the adsorbed hemoglobin was easily eluted with a recovery rate of 86.8%. In addition, Cu-PDA-UTMS shows good reusability up to six cycles. In the end, the adsorption properties by Cu-PDA-UTMS towards hemoglobin from human blood samples were analyzed by SDS-PAGE. The results showed that Cu-PDA-UTMS are a high-performance IMAC adsorbent for hemoglobin separation, which provides a new method for the effective separation and purification of hemoglobin from complex biological samples.

Molecularly Imprinted Polymers with Shape-Memorable Imprint Cavities for Efficient Separation of Hemoglobin from Blood.

Efficient separation and purification of hemoglobin from blood and other complicated biological fluids still remains a big challenge. Molecularly imprinted polymers (MIPs) of hemoglobin are potential choices; however, they suffer from severe problems including difficult template removal and low imprinting efficiency like other protein-imprinted polymers. Herein, a novel MIP of bovine hemoglobin (BHb) was designed in which a peptide crosslinker (PC), instead of the commonly used crosslinkers, was used. The PC, a random copolymer of lysine and alanine, adopts an α-helical conformation at pH 10 but transits to a random coil conformation at pH 5. The introduction of alanine residues lowers the pH range at which the PC undergoes helix-coil transition. The imprint cavities in the polymers are shape-memorable due to the reversible and precise helix-coil transition of the peptide segments in the polymers. They can be enlarged by lowering pH from 10 to 5, thus allowing complete removal of the template protein under mild conditions. When the pH is adjusted back to 10, their original size and shape will be recovered. Therefore, the MIP binds the template protein BHb with high affinity. Compared with the MIP crosslinked with the commonly used crosslinker, the imprinting efficiency of the PC-crosslinked MIP is significantly improved. In addition, both the maximum adsorption capacity (641.9 mg/g) and imprinting factor (7.2) are much higher than the BHb MIPs reported previously. The new BHb MIP also exhibits high selectivity toward BHb and good reusability. Thanks to the high adsorption capacity and high selectivity of the MIP, when it was applied to extract BHb from bovine blood, BHb in the blood sample was extracted almost completely, and high purity product was obtained.

Preparation of bacterial cellulose/vinyl imidazole-based membranes for selective purification of Hemoglobin

The development of new biomaterials is needed to use in many areas such as protein purification, removal and drug release system. In this study, we fabricated hemoglobin (Hb) surface imprinting onto the bacterial cellulose nanofibers. The metal ions coordination was obtained with vinyl imidazole and Ni2+ ions. The selective purification of Hb was obtained using Hb-imprinted bacterial cellulose. The Scanning Electron Microscopy, Surface area, Transformed Infrared Spectroscopy, swelling tests and contact angle measurements were used for the characterization of Hb-imprinted and non-imprinted bacterial cellulose membranes. In addition, the adsorption studies were experimented with using the Hb aqueous solution in the batch systems at 2 h. In this study, the prepared bacterial cellulose was reported unique biomaterials for selective and sensitive purification of Hb with high adsorption capacity. The reusability of bacterial celluloses was demonstrated during the experiment.

Interface imprinted polymers with well-oriented recognition sites for selective purification of hemoglobin

In this study, we introduced a new strategy to design interface imprinted polymers for a novel aspect of molecular imprinting technique, utilization of sacrificial metal oxide particles. In the first step, bovine hemoglobin (BHb) was adsorbed on zinc oxide (ZnO) particles, which were then used to synthesize polyacrylic acid-based molecular imprinting membrane by bulk polymerization in the presence of ethylene glycol dimethacrylate as a cross-linking agent. After polymerization terminated, BHb-ZnO particles were removed to leave effective imprint sites onto the bulk polymeric network which is responsible for the formation of template orientation. The characterization of membranes was investigated by using Fourier transform infrared (FTIR), Raman spectroscopy (RS), scanning electron microscopy (SEM), surface area measurements (BET analyses) and thermogravimetric analysis (TGA). The interface molecularly imprinted membranes (iMIMs) have a relatively high specific rebinding capacity of 65.98 mg/g and excellent selectivity towards BHb with a separation factor of 6.78. The equilibrium adsorption isotherms fitted well to Langmuir isotherms (R2 = 0.9944) and the value of adsorption capability (Qmax) and equilibrium constant (b) were estimated to be 73.53 mg/g and 1.36 mg/mL for the iMIM, respectively. The kinetics of adsorption fitted best to pseudo-second order (R2 = 0.9912). The ZnO particles were used not only to ensure the preservation of the imprint cavities in the polymer network but also to lead to high template removal and better rebinding kinetics. This novel design with multiple recognition sites is quite simple and suitable for the separation of biomacromolecules.

Preparation of bottlebrush polymer-modified magnetic graphene as immobilized metal ion affinity adsorbent for purification of hemoglobin from blood samples.

An immobilized metal affinity (IMA) adsorbent was prepared by grafting bottlebrush polymer pendant with iminodiacetic acid (IDA) from the surface of polydopamine (PDA)-coated magnetic graphene oxide (magGO), via surface-initiated atom transfer radical polymerization (SI-ATRP). Poly(hydroxyethyl methacrylate) (PHEMA) was grafted firstly from the PDA-coated magGO as the backbone, and then poly(glycidyl methacrylate) was grafted from the PHEMA chains via the second SI-ATRP to afford the bottlebrush polymer-grafted magGO Thereafter,IDA was anchored on the nanocomposites to produce the IMA adsorbent after chelating copper ions. The adsorbent was characterized by various physical and physicochemical methods. Its adsorption properties were evaluated by using histidine-rich proteins (bovine hemoglobin, BHb) and other proteins (lysozyme and cytochrome-C). The results show that its maximum adsorption capacity to BHb was 378.6mgg-1, and the adsorption equilibrium can be quickly reached within 1h. The adsorbent has excellent reproducibility and reusability. It has beenapplied to selectively purify hemoglobin from human whole blood, indicating its potential in practical applications. Graphical abstract.

Purification of hemoglobin by adsorption on nitrogen-doped flower-like carbon superstructures.

Nitrogen-doped flower-like carbon superstructures (NPC-F) are prepared via carbonizing self-assembled polyimide nanosheets. SEM, TEM, XPS, and N2 sorption methods are adopted to characterize the flower-like structure. NPC-F exhibits adsorption selectivity for hemoglobin (Hb) because the specific pyridinic N groups of NPC-F could coordinate with the sixth vacancy of ferrous ion in hemoglobin. The adsorption behavior fits well with Langmuir model with a maximum adsorption capacity of 360.0mgg-1 and the adsorbed Hb could be lightly stripped from the NPC-F nanospheres surface by 0.5wt% CTAB solution. Circular dichroism spectra indicate no obvious conformation changing of Hb during purification process by NPC-F nanospheres. Five cycles of a continuous adsorption/desorption experiment demonstrate the reusability of NPC-F as adsorbent for Hb. The prepared NPC-F superstructures are then employed for the isolation of Hb from human whole blood sample, obtaining high-purity Hb as demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis assays. Graphical abstractNitrogen-doped flower-like carbon superstructure (NPC-F) is used to isolate target protein. NPC-F exhibits highly selective capture capacity towards hemoglobin because the specific pyridinic N groups of NPC-F could coordinate with the sixth vacant coordinating position of Fe2+ in hemoglobin.

A novel lanthanide-chelate based molecularly imprinted cryogel for purification of hemoglobin from blood serum: An alternative method for thalassemia diagnosis

Purification and analyzing of proteins is an essential means for understanding their function and diseases associated with their lack or defect. In this research, a new lanthanide-chelate based molecularly imprinted polymer (MIP) was synthesized for selective separation of Hemoglobin (Hb) from human serum in the presence of various interference molecules. The Hb-imprinted polymer was prepared by using complex functional monomer N-methacryloylamido antipyrine (MAAP)-Ce(III) and 2-Hydroxyethyl methacrylate (HEMA) in accordance of cryopolymerization techniques. The nonimprinted cryogel (NIP) was also prepared at same polymerization conditions in the absence of template Hb molecule. The effects of pH, initial Hb concentration, flow rate, temperature and ionic strength on the binding capacity of both imprinted and nonimprinted cryogels was investigated. The maximum binding capacity for the MIP column was found to be as 79.41 mg g−1 dry cryogel, that is four times higher than the NIP column under the optimum conditions (pH 5.0, flow rate: 1.0 mL min−1, T: 25 °C). Moreover, selectivity experiments were performed by using two interference proteins as myoglobin (Mb) and cytochrome c (Cyt-c) and the relative selectivity coefficients (k') for Hb/Mb and Hb/Cyt-c pairs were determined as 36.59 and 37.22, respectively.

Ni(II)-decorated porous titania microspheres as a stationary phase for column chromatography applications: Highly selective purification of hemoglobin from human blood

Titania (TiO2)-based monodisperse-porous stationary phase/sorbent was synthesized by decoration of Ni(II) ions onto TiO2 microspheres 4.2 µm in size, obtained by a staged-shape template hydrolysis and condensation protocol. Ni(II) ions were attached onto iminodiacetic acid-3-glycidoxypropyltrimethoxysilane (IDA-GPTMS) bound-titania microspheres by metal-chelate complex formation. The appropriate mean size, sufficiently high surface area and high porosity providing an appropriate column permeability make Ni(II)-decorated TiO2 microspheres a good sorbent/stationary phase for batch/continuous-column chromatography applications. Ni(II)-decorated TiO2 microspheres were investigated as a sorbent for purification of a typical histidine-rich protein, hemoglobin (Hb) via immobilized metal affinity chromatography (IMAC) in batch fashion, by including bovine serum albumin (BSA) as reference. The saturation capacities of batch adsorption runs performed with bovine Hb and BSA were determined as 137 ± 9 and 45 ± 3 mg/g, respectively. Human Hb with the purity of > 95% was recovered from whole blood by IMAC conducted in batch-fashion. Ni(II)-decorated microspheres were also evaluated as a stationary phase in a microfluidic-IMAC system, in which, human Hb was recovered from whole blood with a purity of 85%. The microfluidic-IMAC system constructed here, based on monodisperse-porous TiO2 microspheres, is a promising tool for genomics/proteomics applications involving isolation of valuable biomolecules from low-volume samples.

Composite imprinted macroporous hydrogels for haemoglobin purification from cell homogenate

Purification of haemoglobin (Hb) has been studied for many years due to its ability to act as an oxygen carrier and its possible use in urgent clinical treatment. In this study, different types of chromatography columns were developed for Hb purification. Two of them showed satisfactory results as affinity chromatography columns and were thus studied more extensively. The affinity adsorbents were prepared by molecular imprinting techniques. In the first case, Pickering emulsion polymerization was used to prepare affinity adsorbents based on molecular imprinting technology. The imprinted particles were immobilized via covalent bonds on the surface of cryogel, a macroporous hydrogel produced by free radical polymerization under sub-zero temperature. In the second case, the affinity sites for Hb were formed directly on an acrylamide cryogel by protein imprinting during the cryogelation. The dynamic binding capacity of the composite cryogel with the immobilized particles and the directly imprinted acrylamide cryogel was found to be 5.2 mg/g and 3.6 mg/g, respectively. The affinity columns showed high selectivity towards Hb in spite of the presence of serum albumin as well as other interfering substances in non-clarified cell homogenates. The maximum capacity in batch mode, the fluid flow and other physical and chemical properties of these columns were investigated.

Surface imprinted bacterial cellulose nanofibers for hemoglobin purification

There is a significant need for the development of the novel adsorbents in the field of protein purification. In this study, thin hemoglobin imprinted film (MIP) was fabricated onto the bacterial cellulose nanofibers’ (BCNFs) by surface imprinting method using metal ion coordination interactions with N-methacryloyl–(L)-histidinemethylester (MAH) and copper ions. The hemoglobin surface imprinted bacterial cellulose nanofibers (MIP-BCNFs) was applied to selective recognition of hemoglobin and purification from hemolysate. The characterization of the MIP-BCNFs was carried out by the Fourier Transformed Infrared Spectroscopy (FT-IR), Scanning Electron Microscopy (SEM), micro-Computerized Tomography (μCT), atomic force microscopy (AFM) and surface area measurements. The adsorption experiments of hemoglobin onto the MIP-BCNFs and NIP-BCNFs from aqueous hemoglobin solutions were investigated in a batch system. The results showed that MIP-BCNFs are promising materials for purification of hemoglobin with high adsorption capacity, significant selectivity and reusability.

The relationship between extent of hemoglobin purification and the performance characteristics of a blood-based flocculant.

Whole blood is a highly complex substance. Hemoglobin, the most abundant blood protein, can function as a flocculant; most of the other blood components exhibit poor flocculant activity. For the purpose of processing raw whole blood into a flocculant product, the practical value of hemoglobin purification is uncertain. This study compares the flocculant performance of whole blood to that of three different semi-purified hemoglobin preparations. The whole blood is processed to remove the plasma proteins, the solid cell components, or both. The flocculant performance of whole blood and each hemoglobin preparation is compared over wide ranges of flocculant dose and suspension pH. The clarified liquids are examined for increases in chemical oxygen demand and Kjeldahl nitrogen. Hemoglobin preparations that excluded plasma gave peak flocculation performance at approximately 30 mg solids per gram of suspended kaolin, and gave greatly reduced performance at higher doses; preparations that included plasma gave very similar peak performance, but also maintained relatively high performance at doses up to at least 200 mg g-1 . It is shown that removal of the plasma and the cell solids does not improve the flocculant performance or lessen the residual pollutants in the treated water. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

Impact of glutathione-HbA1c on HbA1c measurement in diabetes diagnosis via array isoelectric focusing, liquid chromatography, mass spectrometry and ELISA

Hemoglobin A1c (HbA1c) has been proven to be a key biomarker for diabetes screening, and glutathiolation of HbA1c (viz., GSS-HbA1c) has been identified. However, the impact of GSS-HbA1c on the measurement of HbA1c for diabetes screening has not been quantitatively assessed yet. To address the issue, the micropreparative capillary isoelectric focusing (cIEF) developed in our previous work was used for the high resolution separation and purification of hemoglobin (Hb) species. The main fractions of HbA0, HbA3 and HbA1c extracted from the developed cIEF were identified by validated Mono S method. The proposed GSS-HbA1c fractions in the cIEF were pooled and identified by electrospray ionization mass spectrometry (ESI-MS). The HbA1c enzyme-linked immunosorbent assay (ELISA) kit was employed for further quantitative analysis of GSS-HbA1c. A total of 34 blood samples with HbA1c levels from 4.2% to 13.4% were assessed via the above comprehensive strategy of IEF-HPLC-MS-ELISA. It was demonstrated that the HbA1c levels detected by cation exchange LC were considerably influenced by the glutathiolation of Hb and the range of detected GSS-HbA1c values was between 0.23% and 0.74%. The results and developed cIEF methods have considerable significances for investigation of diabetes and clinical diagnosis.

Purification of hemoglobin from red blood cells using tangential flow filtration and immobilized metal ion affinity chromatography

Two methods for purifying hemoglobin (Hb) from red blood cells (RBCs) are compared. In the first method, red blood cell lysate is clarified with a 50nm tangential flow filter and hemoglobin is purified using immobilized metal ion affinity chromatography (IMAC). In the second method, RBC lysate is processed with 50nm, 500kDa, and 50–100kDa tangential flow filters, then hemoglobin is purified with IMAC. Our results show that the hemoglobins from both processes produce identical Hb products that are ultrapure and retain their biophysical properties (except for chicken hemoglobin, which shows erratic oxygen binding behavior after purification). Therefore, the most efficient method for Hb purification appears to be clarification with a 50nm tangential flow filter, followed by purification with IMAC, and sample concentration/polishing on a 10–50kDa tangential flow filter.

Purification of hemoglobin by tangential flow filtration with diafiltration

A recent study by Palmer, Sun, and Harris (Biotechnol. Prog., 25:189-199, 2009) demonstrated that tangential flow filtration (TFF) can be used to produce HPLC-grade bovine and human hemoglobin (Hb). In this current study, we assessed the quality of bovine Hb (bHb) purified by introducing a 10 L batch-mode diafiltration step to the previously mentioned TFF Hb purification process. The bHb was purified from bovine red blood cells (RBCs) by filtering clarified RBC lysate through 50 nm (stage I) and 500 kDa (stage II) hollow fiber (HF) membranes. The filtrate was then passed through a 100 kDa (stage III) HF membrane with or without an additional 10 L diafiltration step to potentially remove additional small molecular weight impurities. Protein assays, SDS-PAGE, and LC-MS of the purified bHb (stage III retentate) reveal that addition of a diafiltration step has no effect on bHb purity or yield; however, it does increase the methemoglobin level and oxygen affinity of purified bHb. Therefore, we conclude that no additional benefit is gained from diafiltration at stage III and a three stage TFF process is sufficient to produce HPLC-grade bHb.

Polyethylene glycol increases purification and recovery, alters retention behavior in flow-through chromatography of hemoglobin

Flow-through chromatography was a method for purification of hemoglobin (Hb) from red cell lysate. The presence of 0–5% polyethylene glycol (PEG) increased the retention time of Hb peak from 15min to 20min in flow-through ion-exchange chromatography (IEC) but decreased the retention time from 88min to 62min in the hydrophobic interaction chromatography (HIC). However, the purification and the recovery were both increased. For IEC the recovery of hemoglobin increased from 75% to more than 90%, and the purified Hb showed single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and one peak in size-exclusion HPLC. For HIC, the recovery of hemoglobin was improved significantly from 20% to 85% and the removal of lipids was 100%. The bioactivity of hemoglobin was well preserved in these two chromatographic processes. The mechanism for the effect of PEG in these two flow-through chromatographic processes was discussed.

Purification and characterization of human hemoglobin: effect of the hemolysis conditions

Human hemoglobin was isolated and purified by anion exchange chromatography. To isolate hemoglobin, outdated red blood cells (RBC) were transformed into carbonylhemoglobin, by reaction with carbon monoxide, and submitted to washing/centrifugation procedures, to eliminate other plasma proteins. Albumin was quantified in each supernatant, by the bromcresol green method. Hemolysis was performed in three different hypotonic media (water, 0.01 M NaCl and 5 mM Tris/HCl buffer at pH 7.4), at 8 °C for 24 h. Sonication for 5 min was also used to lyse RBC. After isolation of hemoglobin, additional purification was carried out by anion exchange chromatography on AG MP-1, Q-SFF and both exchangers. Hemoglobin concentration of hemolysates and of purified solutions were determined by the hemiglobincyanide method. Residual phospholipids were extracted from the four different hemolysates, as well as from the purified hemoglobin solutions, and were analyzed by high performance liquid chromatography. Native and SDS-polyacrylamide gel electrophoresis experiments were performed on purified hemoglobin samples to verify the presence of proteins other than hemoglobin. According to the results, the hemolysis conditions have influence on the purification of hemoglobin.

Cloning, expression, purification, and preliminary characterization of a putative hemoglobin from the cyanobacterium Synechocystis sp. PCC 6803.

The genome of the unicellular cyanobacterium Synechocystis sp. PCC 6803 contains a gene (slr2097, glbN) encoding a 123 amino-acid product with sequence similarity to globins. Related proteins from cyanobacteria, ciliates, and green algae bind oxygen and have a pronounced tendency to coordinate the heme iron with two protein ligands. To study the structural and functional properties of Synechocystis sp. PCC 6803 hemoglobin, slr2097 was cloned and overexpressed in Escherichia coli. Purification of the hemoglobin was performed after addition of hemin to the clarified cell lysate. Recombinant, heme-reconstituted ferric Synechocystis sp. PCC 6803 hemoglobin was found to be a stable helical protein, soluble to concentrations higher than 500 microM. At neutral pH, it yielded an electronic absorption spectrum typical of a low-spin ferric species, with maxima at 410 and 546 nm. The proton NMR spectrum revealed sharp lines spread over a chemical shift window narrower than 40 ppm, in support of low-spin hexacoordination of the heme iron. Nuclear Overhauser effects demonstrated that the heme is inserted in the protein matrix to produce one major equilibrium form. Addition of dithionite resulted in an absorption spectrum with maxima at 426, 528, and 560 nm. This reduced form appeared capable of carbon monoxide binding. Optical data also suggested that cyanide ions could bind to the heme in the ferric state. The spectral properties of the putative Synechocystis sp. PCC 6803 hemoglobin confirmed that it can be used for further studies of an ancient hemoprotein structure.

5545328 Purification of hemoglobin by displacement chromatography : Pliura Diana; Wiffen Diane; Ashraf Salman; Magnin Anthony Mississauga, Canada Assigned to Hemosol Inc

5545328 Purification of hemoglobin by displacement chromatography : Pliura Diana; Wiffen Diane; Ashraf Salman; Magnin Anthony Mississauga, Canada Assigned to Hemosol Inc

Purification of Hemoglobin from the Actinorhizal Root Nodules of Myrica gale L.

Hemoglobins are generally absent or present in low concentrations in the nodules of actinorhizal plants. An exception is Casuarina, where a hemoglobin occurs at relatively high concentration. However, this plant is unique in that Frankia, the microsymbiont, lacks the vesicles that are normally the site of nitrogen fixation. The present paper shows that a hemoglobin also occurs at high concentrations in Myrica gale L., an actinorhizal plant in which Frankia does form vesicles. Hemoglobin was extracted from root nodules under anaerobic conditions using a buffer containing CO, detergent, and a reducing agent. Carboxyhemoglobin was purified using gel filtration followed by aerobic ion-exchange chromatography. The optical absorption spectra of the oxy-, deoxy-, and carboxyhemoglobins were similar to those of other hemoglobins. The molecular mass of the native hemoglobin estimated by gel filtration was 38,500 D. The molecular mass of the subunits estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 16,200 D, consistent with the mass of other hemoglobin subunits. Thus, the native hemoglobin is probably a dimer.

Copper Induced Polymerization of Hemoglobin from the Ocean Pout, Macrozoarces americanus

Hemoglobins that polymerize are not uncommon among amphibians, reptiles, and elasmobranchs, but are rare among teleosts and humans. They are useful models for the study of protein structure, function, and molecular evolutionary relationships. The freshwater teleost Hoplias malabarica (I), the marine teleost Lophius americanus (2), and the clown fish, Amphiprion (3) are the only bony fishes reported to have hemoglobins that polymerize. Hoplias and Lophius polymerize by forming intermolecular disulfide bonds, while the polymerization mechanism for the clown fish is, as yet, unknown. Among humans, hemoglobins Ta-Li, Mississippi, and Pot-to Alegre (4, 5, 6) have a single mutation involving the substitution of a cysteine residue at different loci on the globin chain. We now report that the ocean pout has a single hemoglobin with six sullhydryl groups per tetramer and polymerizes, in vitro, after oxidation with potassium ferricyanide (K,Fe(CN),) or cupric chloride (CuClJ. Procedures for hemoglobin purification by DE-52 cellulose chromatography, molecular weight estimations by gel filtration on G100 columns, and urea gel electrophoresis for globin subunit analysis have been described (2). Hemoglobin was incubated with a twoto threefold molar excess of CuCIZ for 15 min in an ice bucket. The excess copper was removed by dialysis or by gel filtration from Sephadex G-25 columns equilibrated with 0.1 M K-PO., buffer, pH 7.4. Thereafter 20-40 mg of the hemoglobin was applied to and eluted from G100 columns. One-ml fractions were collected and the absorbance of each measured at 540 nm. The approximate polymer molecular weight was estimated from the elution volume of the peak tube. Hemoglobin solutions (0.52.0 g/%) were also oxidized by adding l-3 mg of potassium ferricyanide per ml of hemoglobin. Iodoacetamide (IAA)-alkylated hemoglobin ( 15 rmoles/mg hemoglobin) was dialyzed overnight, centrifuged, and subsequently treated with CuC12. In a separate xperiment, a 60-fold molar excess of beta-mercaptoethanol (B-ME) was added to CuCl*-treated hemoglobin, incubated for 1 h, and dialyzed for 3 h before G100 chromatography. Untreated hemoglobin and hemoglobin treated with CuCIZ alone served as controls and were dialyzed for the same period. The gel filtration profile of known molecular weight proteins is shown in Figure 1 A. The inset graph is a plot of the log of the molecular weight of each standard versus the elution volume of the peak tube. The linear relationship indicates that the peak tube elution volume provides a reasonable stimate of the protein molecular weight between 16,000 and 150,000 Da. (The molecular weight fractionation range for Sephadex G-100 is 4 X 10’ and 1.5 X 105.) .The effect of CuCl* on pout hemoglobin is shown in Figure 1 B. In the absence of the cation, the peak tube eluted at a volume comparable to human hemoglobin, suggesting anormal molecular weight for pout hemoglobin of approximately 60,000. The addition of CuC12 results in a polymer with a minimum molecular mass of 100 to 155 kDa, which corresponds to aggregates of, perhaps, two to three tetramers. Cupric-chloride-induced polymer formation may be inhibited by prior incubation with IAA (Fig. 1C). Hemoglobin alkylated with IAA elutes at the same volume as normal pout and human hemoglobin, but in the absence of IAA, cupric-chloride-induced