Purification of bovine hemoglobin via fast performance liquid chromatography

Bovine hemoglobin (bHb) was purified from bovine red blood cells (bRBCs) via anion exchange chromatography preceded by dialysis. This is a fast and effective way to obtain bHb from bRBCs using Q Sepharose XL, a strong anion exchange resin. This resin had double the binding capacity for bHb compared to three other anion exchange resins that were studied in this work. Methemoglobin levels remained below 2% with bHb concentrations between 0.7 and 1.7 mM. The high purity of bHb was confirmed via SDS-PAGE and size exclusion chromatography (SEC).     

Biochemical Characterization of Hemoglobins from Caspian Sea Sturgeons (<Emphasis Type="Italic">Acipenser persicus and Acipenser stellatus</Emphasis>)

Hemoglobin (Hb) variability is a commonly used index of phylogenetic differentiation and molecular adaptation in fish enabling them to adapt to different ecological conditions. In this study, the characteristics of Hbs from two Sturgeon species of the Southern Caspian Sea Basin were investigated. After extraction and separation of hemoglobin from whole blood, the polyacrylamide gel electrophoresis (SDS-PAGE), cellulose acetate electrophoresis, and isoelectric focusing (IEF) were used to confirm Hb variabilities in these fishes. We showed that although both species have variable Hbs with different isoelectric points, their dominant Hbs can be identified. Ion exchange on CM-cellulose chromatography was used for purification of the dominant Hbs from these fishes. The accuracy of the methods was confirmed by IEF and SDS-PAGE. Spectral studies using fluorescence spectrophotometery indicated that although the Hbs from these fishes had similar properties they exhibited clear differences with human Hb. A comparative study of Hbs alpha-helix secondary substructures was performed by circular dichroism spectropolarimetry (CD) analysis. UV–vis spectrophotometery was also utilized to measure oxygen affinity of Hbs by sodium dithionite. Oxygen affinities of these Hbs were compared using Hb–oxygen dissociation curves. Together, these results demonstrate a significant relationship between oxygen affinity of fish hemoglobins and environmental partial pressure of oxygen.     

On-Column Refolding and Purification of Recombinant Human Interleukin-1 Receptor Antagonist (rHuIL-1ra) Expressed as Inclusion Body in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Recombinant human interleukin-1 receptor antagonist (rHuIL-1ra) was produced in E. coli as an inclusion body. rHuIL-1ra was purified to Over 98% purity by anion exchange chromatography after on-column refolding. The optimized processes produced more than 2 g pure refolded rHuIL-1ra per 1 l culture, corresponding to a 44% recovery, without an intermediate dialysis step. Refolded rHuIL-1ra had full biological activity with the MTT assay. An intramolecular disulfide linkage in the oxidized recombinant protein was suggested by data from HPLC and non-reducing SDS-PAGE.     

Separation of hemoglobin types by cation-exchange high-performance liquid chromatography

The use of a recently developed cation-exchange HPLC packing material for the separation of hemoglobin types in human blood has been investigated. Adult and newborn hemolysates from normal individuals and from subjects with hemoglobin disorders were analyzed using a weak cation carboxymethyl-bonded phase on 5-micron-particle-size silica. Elution was accomplished using a Bistris (2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1, 3-propanediol) gradient. Seven well-resolved HbA1 fractions eluted before the major HbA peak. Hbs A1a, A1b, A1c and an HbA1 fraction that increased with aging of the hemolysates were separately eluted. HbF when present or when added to the hemolysates eluted as a distinct peak. HbA was followed by Hbs A2, S, and C when present. An early-eluting peak corresponding to Hb Bart's was identified in newborn hemolysates. It is concluded that cation-exchange HPLC provides a new tool for the reliable separation of minor hemoglobin components.     

Tangential flow filtration facilitated fractionation and PEGylation of low and high-molecular weight polymerized hemoglobins and their biophysical properties

Various types of hemoglobin (Hb)-based oxygen carriers (HBOCs) have been developed as red blood cell substitutes for treating blood loss when blood is not available. Among those HBOCs, glutaraldehyde polymerized Hbs have attracted significant attention due to their facile synthetic route, and ability to expand the blood volume and deliver oxygen. Hemopure®, Oxyglobin®, and PolyHeme® are the most well-known commercially developed glutaraldehyde polymerized Hbs. Unfortunately, only Oxyglobin® was approved by the FDA for veterinary use in the United States, while Hemopure® and PolyHeme® failed phase III clinical trials due to their ability to extravasate from the blood volume into the tissue space which facilitated nitric oxide scavenging and tissue deposition of iron, which elicited vasoconstriction, hypertension and oxidative tissue injury. Fortunately, conjugation of poly (ethylene glycol) (PEG) on the surface of Hb is capable of reducing the vasoactivity of Hb by creating a hydration layer surrounding the Hb molecule, which increases its hydrodynamic diameter and reduces tissue extravasation. Several commercial PEGylated Hbs (MP4®, Sanguinate®, Euro-PEG-Hb) have been developed for clinical use with a longer circulatory half-life and improved safety compared to Hb. However, all of these commercial products exhibited relatively high oxygen affinity compared to Hb, which limited their clinical use. To dually address the limitations of prior generations of polymerized and PEGylated Hbs, this current study describes the PEGylation of polymerized bovine Hb (PEG-PolybHb) in both the tense (T) and relaxed (R) quaternary state via thiol-maleimide chemistry to produce an HBOC with low or high oxygen affinity. The biophysical properties of PEG-PolybHb were measured and compared with those of commercial polymerized and PEGylated HBOCs. T-state PEG-PolybHb possessed higher hydrodynamic volume and P₅₀ than previous generations of commercial PEGylated Hbs. Both T- and R-state PEG-PolybHb exhibited significantly lower haptoglobin binding rates than the precursor PolybHb, indicating potentially reduced clearance by CD163 + monocytes and macrophages. Thus, T-state PEG-PolybHb is expected to function as a promising HBOC due to its low oxygen affinity and enhanced stealth properties afforded by the PEG hydration shell.     

一种安全高效的血红蛋白纯化方法

目的:建立一种适用于大量制备的,安全、高效的血红蛋白纯化方法。方法: 将压积红细胞装入透析袋,以含有还原剂的Tris缓冲液透析破碎,破碎的上清经两级硫酸铵沉淀后透析至上样缓冲体系,离心后取上清即得血红蛋白提取液;红细胞提取液通过阴离子交换柱层析进一步分离,计算回收率。纯化产物浓缩后以SDS-PAGE及HPLC鉴定纯度,进行紫外-可见光谱扫描并以ABL800血气分析仪分析血气指标,以鲎试剂测定内毒素含量,以磷测定法测定脂质含量。结果: 血红蛋白提取液中脂质去除率98%,容易通过0.45μm滤膜;经阴离子交换层析纯化的血红蛋白经SDS-PAGE(银染法)及WB分析没有杂蛋白条带,HPLC分析纯度>99%、总回收率>85%;内毒素含量<2 EU,高铁血红蛋白含量<5%。结论: 该血红蛋白纯化方法安全高效、成本低廉、易于放大生产,具有较好的应用前景。     

Encapsulation of hemoglobin inside liposomes surface conjugated with poly(ethylene glycol) attenuates their reactions with gaseous ligands and regulates nitric oxide dependent vasodilation

Acellular hemoglobin (Hb)-based O2 carriers (HBOCs) are being investigated as red blood cell (RBC) substitutes for use in transfusion medicine. However, commercial acellular HBOCs elicit both vasoconstriction and systemic hypertension which hampers their clinical use. In this study, it is hypothesized that encapsulation of Hb inside the aqueous core of liposomes should regulate the rates of NO dioxygenation and O2 release, which should in turn regulate its vasoactivity. To test this hypothesis, poly(ethylene glycol) (PEG) conjugated liposome-encapsulated Hb (PEG-LEHs) dispersions were prepared using human and bovine Hb. In this study, the rate constants for O2 dissociation, CO association, and NO dioxygenation were measured for free Hb and PEG-LEH dispersions using stopped-flow UV-visible spectroscopy, while vasoactivity was assessed in rat aortic ring strips using both endogenous and exogenous sources of NO. It was observed that PEG-LEH dispersions had lower O2 release and NO dioxygenation rate constants compared with acellular Hbs. However, no difference was observed in the CO association rate constants between free Hb and PEG-LEH dispersions. Furthermore, it was observed that Hb encapsulation inside vesicles prevented Hb dependent inhibition of NO-mediated vasodilation. In addition, the magnitude of the vasoconstrictive effects of Hb and PEG-LEH dispersions correlated with their respective rates of NO dioxygenation and O2 release. Overall, this study emphasizes the pivotal role Hb encapsulation plays in regulating gaseous ligand binding/release kinetics and the vasoactivity of Hb.     

Rapid fractionation of bovine transferrin using immobilized gangliosides.

Bovine transferrin (BTF) was fractionated from bovine whey using ganglioside affinity chromatography. After loading the immobilized matrix with a 2% whey solution, the matrix was washed with sodium acetate buffer at pH 4 containing 1 M NaCl before elution of BTF with sodium phosphate buffers at pH 7. Concanavalin-A affinity and ion exchange chromatography were used for further purification. The ganglioside column showed a 74.2% BTF recovery from whey and BTF was enriched to 61% purity with ion exchange chromatography. Bovine transferrin was identified by SDS-PAGE and western analysis. The Concanavalin-A affinity and ion exchange chromatography steps enriched BTF in the samples and removed other whey proteins from ganglioside purified fractions. These results indicate that immobilized ganglioside can be used to fractionate BTF from bovine whey. Our novel ganglioside affinity chromatography is rapid and efficient for the fractionation of BTF from whey.     

以聚乙二醇为层析伴侣同时制备SOD、过氧化氢酶和血红蛋白

发展了一条从红细胞裂解液中同时制备超氧化物歧化酶(SOD)、过氧化氢酶和血红蛋白的新工艺。采用0 75 %的聚乙二醇600作为层析伴侣,使血红蛋白直接流过阴离子交换层析柱,同时吸附SOD和过氧化氢酶。经过梯度洗脱获得SOD和过氧化氢酶组分,再经过疏水性相互作用层析与凝胶过滤层析相串联,使SOD和过氧化氢酶得到纯化。纯化后的SOD和过氧化氢酶的比活力分别达到15932u/mg和65918u/mg ,血红蛋白的纯度达到99.9%以上。总回收率为:SOD ,47.4% ;过氧化氢酶,29.6% ;血红蛋白,88.7%。     

Analysis of Biomphalaria glabrata (Gastopoda) hemolymph by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, high-performance liquid chromatography, and immunoblotting

Cell-free hemolymph (serum) of the gastropod mollusc Biomphalaria glabrata was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoreis (SDS-PAGE), high-performance liquid chromatography (HPLC), and immunoblotting. In SDS-PAGE gels, hemoglobin (Hb; apparent molecular weight (aMW) = 160 kDa) was found to be the dominant protein. There were also many subtending polypeptides in the 31,000 to 116,000 aMW range. In order to deplete B. glabrata serum of Hb it was ultracentrifuged by a standard procedure. The supernatant (Hb depleted) and pellet (Hb enriched) were then subjected to SDS-PAGE and HPLC. The polypeptide profile of the Hb-enriched fraction was virtually indistinguishable from whole serum whereas few major proteins were observed in the Hb-depleted fraction. Only by subjecting a large sample to SDS-PAGE followed by the very sensitive silver-staining procedure were several, faintly staining polypeptides observed in the Hb-depleted fraction in the aMW range of 10 to 116 kDa. Similarly, when the Hb-depleted fraction was subjected to HPLC, virtually no peaks were observed. In comparison, several peaks were discriminated from 9 to 12.5 min during chromatography with Hb producing a sharp spike at 13 min. When serum was separated by HPLC and the various fractions were subjected to SDS-PAGE, nearly all samples gave a polypeptide profile similar to whole serum. Rabbits were next used to raise antibodies to whole B. glabrata serum (anti-serum) and the 160-kDa polypeptide (anti-Hb). Rabbit antisera was then tested against whole snail serum and the Hb-depleted and enriched fractions in immunoblot assays. Both antisera gave similar results with reactivity against whole blood and the Hb-enriched fraction being virtually identical. In comparison, both anti-Hb and anti-serum reacted only with the 160-kDa polypeptide as well as two bands at approximately 55 kDa when tested against the Hb-depleted fraction. When the blots were counterstained, no other protein bands were visualized, indicating that each antisera was recognizing all of the proteins present. When the antibodies were tested against serum fractions separated by HPLC, nearly all fractions reacted in a manner similar to whole serum. No other polypeptides were observed when these blots were counterstained. From these experiments, it appears that Hb and Hb subunits comprise the majority of the serum proteins in B. glabrata.     

Hemoglobins from deep-sea hydrothermal vent scaleworms of the genus Branchipolynoe: a new type of quaternary structure

Branchipolynoe symmytilida and B. seepensis are two scaleworms (Polychaeta; Polynoidae) living commensally in the mantle cavity of deep-sea hydrothermal vent and cold-seep mussels. In contrast with littoral members of this family, the two species exhibit a large amount of extracellular hemoglobin (Hb) in their coelomic fluid. Gel filtration revealed the existence of four different Hbs: one minor, high molecular mass (3x10(6) Da) Hb, V1-Hb, reminiscent of a vascular hexagonal bilayer annelid Hb; two major coelomic Hbs, C1-Hb, and C2-Hb, with unusual masses for extracellular annelid Hbs of 153 and 124 kDa respectively; and a minor probably coelomic Hb of 23 kDa (C3-Hb). Using electrospray ionization mass spectrometry, SDS-PAGE after subtilisin treatment, and tandem mass spectrometry, we showed that C1-Hb is a trimer of a 57,996 Da chain and C2-Hb is a dimer of a 57,648 Da chain, each chain being a four-domain/four-heme polypeptide. This multimeric, multidomain arrangement is unique among annelid Hbs and appears different from that of other known multidomain Hbs.     

All hemoglobin-based oxygen carriers are not created equally

Hemoglobin (Hb)-based oxygen carriers (HBOCs) also known as "blood substitutes" have been under active clinical development over the last two decades. Cell-free Hb outside its natural protective red blood cell environment, as is the case with all HBOCs, has been shown to be vasoactive in part due to the scavenging of vascular endothelial nitric oxide (NO) and may in some instances induce heme-mediated oxidative stress. Chemical modification intended to stabilize HBOCs in the tetrameric or polymeric forms introduces conformational constraints that result in proteins with diverse allosteric responses as well as oxidative and nitrosative redox side reactions. Intra and inter-molecular cross-linking may in some instances also determine the interactions between HBOCs and normal oxidative inactivation and clearance mechanisms. Oxygen and oxidative reactions of normal and several cross-linked Hbs as well as their interactions with endogenous plasma protein (haptoglobin) and cellular receptor pathways (macrophage CD163) differ significantly. Therefore, safety and efficacy may be addressed by designing HBOCs with modifications that limit hypertension, minimize heme destabilization and take into account endogenous Hb removal mechanisms to optimize exposure times for a given indication.     

Tangential flow filtration of hemoglobin

Bovine and human hemoglobin (bHb and hHb, respectively) was purified from bovine and human red blood cells via tangential flow filtration (TFF) in four successive stages. TFF is a fast and simple method to purify Hb from RBCs using filtration through hollow fiber (HF) membranes. Most of the Hb was retained in stage III (100 kDa HF membrane) and displayed methemoglobin levels less than 1%, yielding final concentrations of 318 and 300 mg/mL for bHb and hHb, respectively. Purified Hb exhibited much lower endotoxin levels than their respective RBCs. The purity of Hb was initially assessed via SDS‐PAGE, and showed tiny impurity bands for the stage III retentate. The oxygen affinity (P₅₀) and cooperativity coefficient (n) were regressed from the measured oxygen‐RBC/Hb equilibrium curves of RBCs and purified Hb. These results suggest that TFF yielded oxygen affinities of bHb and hHb that are comparable to values in the literature. LC‐MS was used to measure the molecular weight of the alpha (α) and beta (β) globin chains of purified Hb. No impurity peaks were present in the HPLC chromatograms of purified Hb. The mass of the molecular ions corresponding to the α and β globin chains agreed well with the calculated theoretical mass of the α‐ and β‐ globin chains. Taken together, our results demonstrate that HPLC‐grade Hb can be generated via TFF. In general, this method can be more broadly applied to purify Hb from any source of RBCs. This work is significant, since it outlines a simple method for generating Hb for synthesis and/or formulation of Hb‐based oxygen carriers. © 2008 American Institute of Chemical Engineers, 2009     

Site-specific cross-linking of human and bovine hemoglobins differentially alters oxygen binding and redox side reactions producing rhombic heme and heme degradation

Chemically modified human or bovine hemoglobins (Hb) have been developed as oxygen-carrying therapeutics and are currently under clinical evaluation. Oxidative processes, which are in many cases enhanced when modifications are introduced that lower the oxygen affinity, can limit the safety of these proteins. We have carried out a systematic evaluation of two modified human Hbs (O-R-polyHbA(0) and DBBF-Hb) and one bovine Hb (polyHbBv). We have both measured the oxidative products present in the Hb preparations and followed the oxidative reactions during 37 degrees C incubations. Autoxidation, the primary oxidative reaction which initiates the oxidative cascade, is highly correlated with P(50) (R = 0.987; p < 0.002). However, when the results for the other oxidative processes are compared, two different classes of oxidative reactions are identified. The formation of oxyferrylHb, like the rate of autoxidation, increases for all modified Hbs. However, the subsequent reactions, which lead to heme damage and eventually heme degradation, are enhanced for the modified human Hbs but are actually suppressed for bovine-modified Hbs. The rhombic heme measured by electron paramagnetic resonance, which is the initial step that causes irreversible damage to the heme, is found to be a reliable measure of the stability of ferrylHb and has the tendency to produce degradation products. DBBF-Hb, a Hb-based oxygen carrier (HBOC) for which toxic side effects have been well documented, has the highest level of rhombic heme (41-fold greater than for HbA(0)), even though its rate of autoxidation is relatively low. These findings establish the importance of these secondary oxidative reactions over autoxidation in evaluating the toxicity of HBOCs.     

Electrophoretic and chromatographic evidence for allelic polymorphisms in the river buffalo α-globin gene complex

Isoelectric focusing in the ultranarrow immobilized (7.1–7.5) pH gradient (IPG) of hemoglobin and high-performances liquid chromatography (HPLC) of globin chains were used to investigate Hb polymorphism in Italian river buffalo. Six different phenotypes, each characterized by two or four different Hbs, were detected by IPG, whereas two different^IIα-globin chains were separated from two different^Iα-chains by HPLC. Two α-chains (^Iα¹ and^IIα³), and Hbs with similar mobilities (Hb1 andHb3), were associated with the AA Hb phenotype: two α-chains (^Iα² and^IIα⁴), and Hbs with different mobilities (Hb2 andHb4), were associated with the BB phenotype: two sets of doublet Hbs were associated with the AB phenotype, thus suggesting allelic polymorphisms at the two α loci. An allele at the β locus is responsible for increasing to as many as eight the number of different Hbs, thus further complicating the notable Hb polymorphism of the river buffalo.     

Functional comparison of hemoglobin purified by different methods and their biophysical implications

Hemoglobin (Hb) that is purified from red blood cells (RBCs) is commonly subjected to harsh processing conditions, such as high temperatures and extensive column separation, which may damage the Hb by altering the heme prosthetic group and/or the Hb protein structure. In this study, bovine and human Hb purified by tangential flow filtration (TFF) was compared to commercial preparations of human Hb (Hemosol, Inc., Toronto, Canada) and bovine Hb (Biopure, Inc., Cambridge, MA). Purified Hbs were characterized by measuring their overall purity (SDS–PAGE, SEC, and ESI‐MS), susceptibility to oxidation (k_ox), responses to physiological conditions (pH, [Cl^?], [IHP], and T), and ligand binding kinetics (O₂, NO, and CO). All Hbs evaluated possessed comparable biophysical properties, however, a small amount of catalase was detected in the TFF‐purified Hbs that reduced the rate of autoxidation. Mass changes observed by mass spectrometry suggest that structural alterations may be introduced into Hbs that are purified by extensive chromatographic separations. These results demonstrate that TFF is a suitable process for the purification of Hb from RBCs with a quality equivalent to that of commercial Hb preparations that employ more extensive purification strategies. This work also shows that TFF can yield highly pure Hb which can be used for Hb‐based O₂ carrier synthesis. Biotechnol. Bioeng. 2010; 106: 76–85. © 2010 Wiley Periodicals, Inc.     

Allosteric effects on oxidative and nitrosative reactions of cell-free hemoglobins

A review of the oxidative and nitrosative reactions of cell-free hemoglobin-based oxygen carriers (HBOCs) shows that these reactions are intimately linked and are subject to allosteric control. Cross-linking reactions used to produce HBOCs introduce conformational constraints and result in Hbs with reduced responses to heterotropic and homotropic allosteric effectors. The Nernst plots of heme oxidation of cross-linked HBOCs are shifted to higher potentials relative to unmodified Hb in the absence of allosteric effectors, in accord with their T-state stabilization and right-shifted Hill plots of O(2) binding. They exhibit enhanced rates of autoxidation and nitrite-induced oxidation, features that appear due to their having more solvent-accessible heme pockets. The stability of their NO-Hb derivatives varies as a result of allosteric effects on the extent of formation of pentacoordinate NO-heme geometry by alpha chains and subsequent oxidation of partner beta chains. The physiological implications of these findings on the safety, efficacy and design of second generation HBOCs are discussed in the framework of a reaction scheme showing linkages between Hb-mediated redox reactions. These redox reactions can drive formation of SNO-Hb and other reactive species and are of significance for the use of cell-free Hbs in vivo.     

Evidence for significant quantities of creatine kinase MM isoenzyme in human brain

Oxygen equilibrium studies have been carried out on hemoglobins A2 (alpha2delta2), Lepore-Washington (alpha2(deltabeta)2) and P-Nilotic (alpha2(beta2delta)2) using the beta chain containing hemoglobins A and S as controls. This investigation was initiated mainly because of controversial data that have been published on the oxygen affinity of hemoglobin (Hb) A2 and because samples containing the rare Hb P-Nilotic became available. Each hemoglobin was isolated in pure form by anion exchange chromatography; the samples used in the equilibrium analyses contained 100 mg Hb/dl with less than 5% ferrihemoglobin and no 2,3--diphosphoglycerate. Oxygen equilibrium analyses were made at 37 degrees C with the method of Benesch et al. (1965) Anal. Biochem. 11, 81--87; Anal. Biochem. 55, 245--248 (1973). A slight, but definite increase in oxygen affinity was observed for Hb A2 as well as for Hb P-Nilotic while the increase for the Hb Lepore-Washington was somewhat greater. The values for n, the Hill coefficient, and the Bohr effects were the same for all hemoglobin types. The differences in oxygen affinity of these hemoglobins apparently result from the differences in primary structure that are characteristic for those proteins.     

Strategies for the purification and characterization of protein scaffolds for the production of hybrid nanobiomaterials

Rotavirus VP6 self-assembles into high order macrostructures useful as novel scaffolds for the construction of multifunctional hybrid nanobiomaterials. This application requires large quantities of high quality pure material with strict structural consistency. Strategies for obtaining high quality recombinant VP6 and different characterization techniques are explored and compared in this work. VP6 was expressed in the insect cell-baculovirus system. VP6 assemblies were selectively purified utilizing an ion exchange and size exclusion (SE) chromatography. Purification steps were monitored and characterized by dynamic light scattering (DLS), ELISA, SDS-PAGE, HPLC and Western blot. DLS showed that the initial ultrafiltration step removed small particles, the intermediate anion exchange chromatographic step completely removed the baculovirus, whereas the final size exclusion chromatography permitted the selective recovery of correctly assembled VP6 nanotubes and discrimination of non-assembled VP6, as confirmed by transmission electron microscopy. VP6 assembled into tubular structures with diameter of 75 nm and several nanometers in length. The purification yield was 20% of multimeric assemblies with a purity >98%. The resulting material was suitable for the production of functionalized hybrid nanobiomaterials through in situ synthesis of metallic nanoparticles.     

Purification and sequence determination of bovine atrial natriuretic factor

We report the purification and the sequence determination of bovine atrial natriuretic factor (ANF) in acid extracts of bovine atrial appendages. The monitoring of the activity along the purification steps was performed with a radio-receptor assay using bovine adrenal cortex membranes sites and 125I ANF. Bovine ANF was separated by carboxymethyl agarose gel chromatography from catecholamines and major protein contaminants. It behaved as a 3 K dalton peptide on Sephadex G-50. The active fractions were then subjected to high performance liquid chromatography (HPLC) using a sulfopropyl cation exchange column. Subsequent purification steps by reverse phase mode on Ultrapore RPSC, Vydac TP 218 and uBondapak using acetonitrile gradients led to the obtention of a pure fraction which amino acid sequence was identical to that for human ANF. This confirms the high degree of homology of ANF structure among mammalian species and advocates the use of the radio-receptor assay based on bovine adrenal receptor for measuring human ANF.