Purification and amino acid analysis of two human monocyte chemoattractants produced by phytohemagglutinin-stimulated human blood mononuclear leukocytes

Physicochemical characteristics of monocyte chemotactic activity in the culture fluid of PHA-stimulated human mononuclear leukocytes (MNL) were investigated. Among several chemotactic activity peaks eluted from a TSK-2000 gel filtration column, one peak, corresponding to a molecular mass of 17 kDa, accounted for about 40% of total chemotactic activity. On a chromatofocusing column, most of the 17-kDa activity eluted in a pH range of 9.4 to 7.9. It could bind to Orange-A Sepharose. These three characteristics--molecular mass, basic isoelectric point, and dye column binding--were similar to those of human glioma-derived monocyte chemotactic factor (GDCF), recently purified in our laboratory. Therefore, the MNL-derived chemoattractant was purified by the same procedures used for purification of GDCF, namely Orange-A Sepharose chromatography, carboxymethyl (CM)-HPLC, and reverse phase (RP) HPLC. About 50% of the culture fluid chemotactic activity bound to Orange-A Sepharose and was eluted in a single peak by a NaCl gradient. The active pool from the Orange-A column was separated into two sharp peaks by CM-HPLC, each of which eluted at identical acetonitrile concentrations from a RP HPLC column. By SDS-PAGE, the peptides had apparent molecular masses of 15 and 13 kDa and appeared homogeneous. Amino acid analysis showed that the composition of the two peptides was almost identical; and the N terminus of each peptide was apparently blocked. Shared characteristics of these peptides and the GDCF peptides include identical elution patterns from CM- and RP HPLC columns, identical SDS-PAGE migration, almost identical amino acid composition, and blocked N terminus. This suggests that the monocyte attractants isolated from culture fluid of PHA-stimulated MNL are identical to those derived from human glioma cells.     

Subunit analysis of bovine cytochrome c oxidase by reverse phase high performance liquid chromatography

Bovine cytochrome c oxidase subunits were separated by reverse phase high performance liquid chromatography using a C4 column eluted with water and an acetonitrile gradient, both containing 0.1% trifluoroacetic acid. Subunits I and III precipitated in this solvent and could not be analyzed; the remaining eleven subunits were dissociated, denatured, soluble and could be resolved by elution from the column. The protein subunit eluting in each chromatographic peak was identified by a combination of polyacrylamide gel electrophoresis in sodium dodecyl sulfate, NH2-terminal amino acid sequencing, and amino acid analysis. Each subunit produced a single elution peak with the exception of subunit VIc (nomenclature of Kadenbach et al., 1983, Anal. Biochem. 129, 517-521), which eluted from the column as two well-resolved peaks. Sequence analysis showed that the two subunit VIc elution peaks resulted from partial chemical blockage of the alpha-amino serine residue of subunit VIc. The C4 reverse phase HPLC was used to document specific subunit removal from bovine cytochrome c oxidase either by tryptic digestion or by dodecyl maltoside extraction. The described HPLC method for separating cytochrome c oxidase subunits should be applicable for the analysis of other multisubunit proteins, especially other multisubunit membrane protein complexes.     

Glycation of lens proteins by the oxidation products of ascorbic acid

Bovine lens water-soluble proteins were incubated with [I-14C]ascorbic acid (ASA) for 6 days, and the incorporation into protein was measured at daily intervals. Aliquots were also withdrawn to determine the distribution of label among the various ASA oxidation products. A linear incorporation into protein was observed in the presence of NaCNBH3, however, little or no incorporation was seen in its absence. TLC analysis showed a complete loss of ASA by day 3, whereas both dehydroascorbate (DHA) and diketogulonic acid (DKG) remained constant for 6 days, consistent with the linear incorporation into protein. The amino acid composition of the proteins glycated in the presence of NaCNBH3 was identical to controls except for a 70% reduction in lysine residues and a corresponding increase in an unknown product which eluted slightly earlier than methionine. In the absence of NaCNBH3 lysine decreased linearly to 20% with an additional decrease in arginine and histidine at later times concurrent with protein crosslinking. DHA and DKG were prepared and incubated directly with lens proteins for an 8 day period. Both compounds glycated lens protein as evidenced by an increased binding to a boronate affinity column. SDS-PAGE showed that both compounds were also capable of causing protein crosslinking. DHA is apparently capable of reacting directly with protein since glycation was observed with the ASA analog, reductic acid, which can be oxidized to dehydroreductic acid, but which cannot be hydrolyzed to an open chain structure. DHA also produced a lysine adduct which was not obtained with DKG, supporting the idea that both species have glycating ability.     

Purification and properties of a pyridoxal 5'-phosphate-dependent histidine decarboxylase from Morganella morganii AM-15

A pyridoxal 5'-phosphate-dependent histidine decarboxylase from Morganella morganii AM-15 was purified to homogeneity. The enzyme is a tetramer (Mr 170,000) of identical subunits and binds 4 pyridoxal-P/tetramer; it is resolved by dialysis against cysteine at pH 6.8. Between pH 6.2 and 8.8, the holoenzyme shows pH-independent absorbance maxima at 333 and 416 nm. Vmax/Km is highest at pH 6.5; this optimum reflects chiefly increased Km values for histidine at lower or higher pH values, whereas Vmax is highest at pH 5.0 and decreases only moderately between pH 5.0 and 8.0. The enzyme also decarboxylates beta-(2-pyridyl)alanine and N tau-methylhistidine (but not N pi-methylhistidine); arginine, lysine, and ornithine are neither substrates nor inhibitors. The hydrazine analogue of histidine, 2-hydrazino-3-(4-imidazolyl)propionic acid, is a very potent competitive inhibitor; other carbonyl reagents and a variety of carboxyl- or amino-substituted histidines also inhibit competitively. alpha-Fluoromethylhistidine is a potent irreversible inhibitor of the enzyme; alpha-methylhistidine is a competitive inhibitor/substrate that is decarboxylated slowly and undergoes a slow decarboxylation-dependent transamination that converts the holoenzyme to pyridoxamine-P and apoenzyme. Dithiothreitol and other simple thiols are mixed-type inhibitors that interact with pyridoxal-P at the active site to form complexes (lambda max congruent to 340 nm), presumably the corresponding thioalkylamines, without resolving the holoenzyme. This histidine decarboxylase (Vmax = 72 mumol X min-1 X mg-1) is much more active than "homogeneous" preparations of mammalian pyridoxal-P-dependent histidine decarboxylase (Vmax congruent to 1.0) and is about equal in activity to the pyruvoyl-dependent histidine decarboxylases from Gram-positive bacteria.     

Protein reactions with methyl and ethyl vinyl sulfones

Disulfide bonds of bovine serum albumin and wool were reduced byn-tributylphosphine to sulfhydryl groups that were then modified by methyl or ethyl vinyl sulfone in a nucleophilic addition reaction toS-(-ethylsulfonylmethyl)-l-cysteine andS(-ethylsulfonylethyl)-l-cysteine, respectively. The reductive alkylation was carried out either simultaneously, with both the reducing and alkylating agents present in the reaction mixture, or sequentially, with the reduced proteins first isolated before alkylation. Amino acid analysis studies showed that authentic, syntheticS-(-ethylsulfonylethyl)-l-cysteine eluted as a well-resolved peak after serine but that the peak associated with the corresponding methyl derivative overlapped the corresponding peak due to threonine. The extent of alkylation of the sulfhydryl groups of cysteine, -NH₂ of lysine, and NH groups of the imidazole ring of histidine was also measured by amino acid analysis. The results show that alkyl vinyl sulfones have a strong chemical affinity for protein functional groups.     

The hepatic glutathione transferases of the male little skate, Raja erinacea

Five cytosolic glutathione transferases were isolated from the liver of the male little skate, Raja erinacea, a marine elasmobranch. They were designated E-1 through E-5 in order of their elution from a DEAE-cellulose column with a 0 to 100 mM KCl gradient in 0.01 M Tris (pH 8.0). Each eluted peak of glutathione transferase activity, after concentration, was applied to an affinity column prepared by reaction of epoxy-activated Sepharose 6B with glutathione (GSH). Elution of the various glutathione transferases from this column with GSH resulted in the further purification of each enzyme; the major glutathione transferase, E-4 and E-1, were purified to apparent homogeneity by this procedure. Skate glutathione transferase E-4 is dimeric and the subunits are either very similar or identical in molecular weight (about 26 000 daltons). Enzymes E-2 through E-5 were acidic proteins (pI less than 7.0) and had high specific glutathione transferase activity (0.3--12 mumol/min/mg protein) with benzo[a]pyrene 4,5-oxide (BPO) as substrate, whereas the other enzyme (E-1) had low activity (0.01 mumol/min/mg) with BPO and a basic pI (greater than 9.5). Bilirubin and hematin, non-substrate ligands, bound tightly to homogeneous E-4, with dissociation constants in the micromolar range.     

Isolation and characterization of an extremely basic protein from adenovirus type 5.

By starch-gel electrophoresis and a staining method that is highly sensitive for argininyl residues, adenovirus type 5 was found to contain two minor basic polypeptides of extreme cathodic mobility in addition to the two known core proteins. The fastest-migrating polypeptide, named mu protein, and the second fastest polypeptide are found in adenovirions and virus-infected KB cells but not in top components or in uninfected cells. The top components and infected cells contain an additional basic polypeptide, presumably P-VII, that migrates slightly slower than polypeptide VII. None of the basic polypeptides of adenovirions was electrophoretically identical to the host histone. The basic proteins of adenovirions were purified by urea phosphocellulose column chromatography and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two minor basic core proteins, mu and another component, have similar mobilities in sodium dodecyl sulfate-polyacrylamide gels as a complex of polypeptides X-XII. After further purification on a Sephadex G-75 column, the mu protein was found to have a molecular weight of about 4,000. Amino acid analysis showed that the mu protein lacks tryptophan and 69% of the total amino acid residues are basic, that is, 54% arginine, 13% histidine, and 2% lysine. Only eight amino acids seem to contribute to make the mu polypeptide. There are 125 copies of the mu polypeptide per 1,000 copies of polypeptide VII in a virion.     

Effective elution of antibodies by arginine and arginine derivatives in affinity column chromatography

It has been shown that the recovery of monomeric antibodies from protein A affinity chromatography is enhanced significantly by using arginine as an eluent. To extend the applications of arginine to antibody purification and obtain an insight into the mechanism of arginine elution, we compared arginine with citrate, guanidine hydrochloride (GdnHCl), arginine derivatives, and other amino acids in protein A chromatography. We also applied arginine to elution of polyclonal antibodies (pAbs) in antigen affinity chromatography. As described previously, arginine was effective in eluting monoclonal antibodies IgG1 and IgG4. Two arginine derivatives, acetyl-arginine and agmatine, resulted in efficient elution at pH 4.0 or higher, and this was comparable to arginine. On the other hand, other amino acids, such as glycine, proline, lysine, and histidine, are much less effective than arginine under identical pH conditions. Whereas elution increased with arginine concentration, elution with citrate was insignificant in excess of 1 M at pH 4.3. Arginine was also effective in fractionation of pAbs using antigen-conjugated affinity columns. Although GdnHCl was also effective under similar conditions, the eluted material showed more aggregation than did the protein eluted by arginine.     

Fractionation of human low density lipoprotein by column chromatography.

We have devised a method to fractionate low density lipoprotein (LDL) into subspecies by means of column chromatography. DEAE-agarose columns, 2.6 X 60 cm, were loaded with LDL (25-45 mg LDL protein) and eluted with a 0.045-0.13 M NaCl gradient. The LDL eluted over a volume of 900 ml. Specific portions of the eluted LDL, reapplied to a column identical with the original, reelute at about the same point. Altering the NaCl concentration of the elution fluid changed the elution volume. The cholesterol-protein ratio of the LDL subfractions was progressively lower in fractions eluting at higher NaCl concentrations. These results indicate the LDL is not a homogenous lipoprotein species but consists of subfractions which differ in at least charge and cholesterol content.     

Displacement effects in large-scale chromatography?

Displacement effects in large-scale (total column volume v(t) = 150 L) and preparative ion-exchange chromatography purifying human erythrocyte superoxide dismutase are described in the present article. The biomolecules are eluted in a very small peak elution volume (<0.2 v(o)) behind the salt wave using a step gradient. The theoretical peak width and retention behavior are calculated according to the model of Yamamoto. The theoretical values are then compared with the experimental data. There was a difference observed between the elution type I (also called fronting) and the experimentally obtained elution. Some instructions are given on how to achieve these phenomenona because a beneficial effect in respect to resolution and recovery of a biomolecule is observed.     

Purification from goat antiserum of immunoglobulins against G6PD from rabbits]

DEAE Affi-Gel Blue (Bio-Rad) provides an efficient and rapid fractionation of human serum proteins by a single chromatographic step. When goat serum is applied to the matrix and chromatography is performed following the procedure utilized for the human serum proteins, the elution pattern changes and the Ig purification is not satisfactory. We achieved a better Ig purification from goat serum by the following improved procedure. We performed first an AS-40 fractionation followed by extensive dialysis in 50 mM Na-citrate pH 5.7. The sample was then loaded onto a P11 column equilibrated in the same buffer. The fraction eluted at Vo contained total IgG and the other serum proteins, except beta-globulins which were eluted with 0.24 M phosphate. Peak 1 concentrated and dialyzed in 20 mM phosphate buffer pH 8 was then applied to a DEAE Affi-Gel Blue column, equilibrated in the same buffer. Two protein peaks were eluted from this column and electrophoretically characterized as: peak 1, containing a pure Ig fraction (70% yield), peak 2 with albumin and other contaminating serum proteins. When goat antiserum is obtained against a specific protein, our technique may be suitably employed to purify polyclonal antibodies for immunoprecipitation studies.     

Strong-cation-exchange sulfoethyl aspartamide chromatography for peptide mapping of Staphylococcus aureus V8 protein digests

In two recent reports (D. L. Crimmins, J. Gorka, R. S. Thoma, and B. D. Schwartz (1988) J. Chromatogr. 443, 63-71; A. J. Alpert and P. C. Andrews (1988) J. Chromatogr. 443, 85-96) a sulfoethyl aspartamide column was shown to efficiently analyze peptides less than 25 residues in length which differ in the number of nominal positive charges at pH 3.0. In particular, the elution order for a series of distinct peptides ranging in nominal charge from +1 to +7 was found to be monotonic in nature indicating that separation was primarily via a cation-exchange mechanism. The present study employs this chromatographic system to isolate and characterize major fragments of proteolytic digests. Six commercially available proteins of known sequence (myoglobin, beta-casein, concanavalin A, carbonic anhydrase, lentil lectin, and enolase) were digested with Staphylococcus aureus V8 to generate peptide fragments. The resulting mixture was chromatographed on a sulfoethyl aspartamide column to isolate major fragments which were then subjected to amino acid analysis and N-terminal sequencing. With complete proteolysis (i.e., peptide fragments terminating in either an aspartic or a glutamic acid) separation of the fragments should result from the sum of histidine, lysine, and arginine residues contained in each fragment. Most of the peptide fragments eluted at the expected time on the sulfoethyl aspartamide column. Those fragments with anomalous behavior resulted from incomplete cleavage or cleavage at nonacidic residues or were greater than 35 residues in length. Each proteolytic digest was also analyzed by standard reverse-phase C4 chromatography to compare the peptide maps for these two distinct chromatographic modes.     

Hemapheresis using vesicular plant separation materials]

The present paper deals with the separation of cells from soluble compounds of blood by means of exclusion chromatography using a recently described vesicular packing material made from the cell wall framework of the small duckweed Wolffia arrhiza. The cells of the periphere blood are hardly retarded in passing through a packing of the vesicular material and eluted as sharp peak at an elution volume which is near to 30% of the column volume. The behavior of cells is similar to that of the excluded high molecular weight plasma proteins (e.g. serumalbumin). Low molecular weight solutes (e.g. salts, glucose, urea, kreatinin), but also substances of considerable molecular weight (e.g. myoglobin and Vitamin B12) which are usually difficult to separate by dialysis from serum, are eluted at nearly 100% of the packing volume and may be separated completely from cells and high molecular weight proteins. In vitro-Tests did not show a reduced vitality of eluted blood cells.     

The structure and mechanism of stem bromelain. Evaluation of the homogeneity of purified stem bromelain, determination of the molecular weight and kinetic analysis of the bromelain-catalysed hydrolysis of N-benzyloxycarbonyl-l-phenylalanyl-l-serine methyl ester

1. Purified stem bromelain (EC 3.4.22.4) was eluted from Sephadex G-100 as a single peak. The specific activity across the elution peak was approximately constant towards p-nitrophenyl hippurate but increased with elution volume with N(2)-benzoyl-l-arginine ethyl ester as substrate. 2. The apparent molecular weight, determined by elution analysis on Sephadex G-100, is 22500+/-1500, an anomalously low value. 3. Purified stem bromelain was eluted from CM-cellulose CM-32 as a single peak and behaved as a single species during column electrophoresis on Sephadex G-100. 4. Purified stem bromelain migrates as a single band during polyacrylamide-gel electrophoresis under a wide variety of conditions. 5. The molecular weight determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate is 28500+/-1000. 6. Sedimentation-velocity and equilibrium-ultracentrifugation experiments, under a variety of conditions, indicate that bromelain is an apparently homogeneous single peptide chain of mol.wt. 28400+/-1400. 7. The N-terminal amino acid composition is 0.64+/-0.04mol of valine and 0.36+/-0.04mol of alanine per mol of enzyme of mol.wt. 28500. (The amino acid recovery of the cyanate N-terminal amino acid analysis was standardized by inclusion of carbamoyl-norleucine at the cyclization stage.) 8. The pH-dependence of the Michaelis parameters of the bromelain-catalysed hydrolysis of N-benzyloxycarbonyl-l-phenylalanyl-l-serine methyl ester was determined. 9. The magnitude and pH-dependence of the Michaelis parameters have been interpreted in terms of the mechanism of the enzyme. 10. The enzyme is able to bind N-benzyloxycarbonyl-l-phenylalanyl-l-serine methyl ester relatively strongly but seems unable to make use of the binding energy to promote catalysis.     

Fast protein separation by reversed-phase high-performance liquid chromatography on octadecylsilyl-bonded nonporous silica gel. II. Improvement in recovery of hydrophobic proteins.

Recovery of hydrophobic proteins from an RP-HPLC column was improved using a fast-separation RP-HPLC system operated at room temperature. Hydrophobic proteins such as ovalbumin could be adequately eluted from a nonporous octadecylsilyl (C18) spherical silica gel with a particle diameter of 20 microns using steep gradient elution with a 0.1% aqueous trifluoroacetic acid-acetonitrile system at a constant flow rate of 4 ml/min. Recoveries improved under fast separation since the protein sample suffered only a slight amount of irreversible denaturation on the hydrophobic surface of the stationary phase. The fast-separation system was also applied to the separation of larger proteins such as apo-ferritin (443 kDa) and thyroglobulin (669 kDa) as well as egg white proteins.     

Studies on the Extracellular Polysaccharides (EPS) Produced in vitro by Pseudomonas phaseolicola

Native EPS produced by Pseudomonas syringae pv. phaseolicola in vitro was separated by ion exchange chromatography on DEAE fractogel into three different polysaccharide fractions. A neutral polysaccharide eluting with the void volume yielded only fructose upon hydrolysis and exhibited an IR spectrum similar to authentic levan. At about 300 mM KCl a mannuronan eluted. Comparison with authentic alginate by IR spectroscopy, elution behaviour during DEAE-fractogel column chromatography, and monomer composition (mannuronic acid and traces of guluronic acid) confirmed the identity of this fraction as a bacterial alginate. It contained about 56 mol% acetyl groups. A third polysaccharide eluted at about 160 mM KCl. Its monomeric composition (rhamnose, fucose, glucose, and amino sugars), elution behaviour upon DEAE-fractogel column chromatography, and TLC patterns, closely resembled the sugar moiety of lipopolysaccharides (LPS) from, Pseudomonas syringae pv. phaseolicola. The protein component of crude EPS represented a fourth macromolecular fraction. It was not covalently linked to any of the polysaccharides since it could be removed from the EPS by phenol extraction.     

Effect of chemical modification on the cryoprecipitation of monoclonal human cryoglobulin M

The effect of the chemical modification of lysine, histidine, arginine, tyrosine, tryptophan residues and carboxylic groups on the cryoproperties of monoclonal human cryoglobulin M has been studied. The modification of 35-40 lysine residues and that of 42-45 arginine residues in the molecule of cryo-IgM has been shown to result in practically complete inhibition of the cryoprecipitation. The same effect is observed on the modification of 60 histidine residues per molecule and on modification of 50 or 51 carboxylic groups. At the same time the modification of practically all the reagent-exposed tryptophan (10 residues per molecule) and tyrosine residues (55 residues per molecule) does not lead to any noticeable decrease in the cryoprecipitation. The conformations of the modified and native proteins are identical according to the circular dichroism data.     

Effects of ionic speciation of lysine on its adsorption and desorption through a sulfone-type ion-exchange column

Lysine produced during microbial fermentation is usually recovered by an ion-exchange process, in which lysine is first converted to the cationic form (by lowering the pH to less than 2.0 with sulfuric acid) and then fed to a cation-exchange column containing an exchanger that has a sulfone group with a weak counterion such as NH4+. Ammonia water with a pH above 11 is then supplied to the column to displace the purified lysine from the column and allow its recovery. To enhance the adsorption capacity and for a possible reduction in chemical consumption, monovalent lysine fed at pH 4 was investigated in comparison with conventional divalent lysine fed at pH 1.5. The adsorption capacity increased by more than 70% on a mass basis using pH 4 feeding compared with pH 1.5 feeding. Lysine adsorbed at pH 4 started to elute earlier than that adsorbed at pH 1.5 when ammonia water was used as the eluant solution, and the extent of early elution became more notable at lower concentrations of ammonia. Moreover, the elution of monovalent lysine fed at pH 4 displayed a stiffer front boundary and higher peak concentration. However, when the ammonium concentration was greater than 2.0 N, complete saturation of the bed was delayed during adsorption and the percent recovery yield from elution was lowered, both drawbacks that were considered inevitable features originating from the increased adsorption of monovalent lysine.     

Purification and some properties of the Glu- and Lys-human plasmin heavy chains.

The heavy polypeptide chains of human Glu-plasmin and human Lys-plasmin have been isolated in native solvents, after partial reduction and carboxymethylation of the corresponding plasmins. Two major forms of each heavy chain can be eluted, after adsorption to Sepharose/lysine, utilizing a gradient of epsilon-aminocaproic acid as the eluant. The elution profile of these heavy chains is practically identical to the elution behavior previously observed for human Glu- and Lys-plasminogen, and human Glu- and Lys-plasmin adsorbed to these columns. Sedimentation velocity analysis of the heavy chain of human Glu-plasmin, in the presence of epsilon-aminocaproic acid, demonstrated that a gross conformational alteration occurs in this peptide accompanying binding of this amino acid. A much smaller conformational alteration occurs under similar circumstances with the human Lys-plasmin heavy chain. We find that the NH2-terminal peptide released in the Glu-plasminogen to Lys-plasminogen and Glu-plasmin to Lys-plasmin conversions is also released in the Glu-plasmin heavy chain to Lys-plasmin heavy chain conversion. This reaction is catalyzed at a significant rate only by plasmin and not by urokinase. Finally, no strong interaction between streptokinase and the isolated plasmin heavy chains is observed.     

Speciation of Al in human serum by convective-interaction media fast-monolithic chromatography with inductively coupled plasma mass spectrometric detection

A new analytical procedure using anion-exchange separation support based on convective-interaction media (CIM) was developed for the speciation of Al in human serum. The separation of proteins was performed on a weak anion-exchange CIM diethylamine (DEAE) fast-monolithic disk. To prevent co-elution of low molecular mass (LMM) Al species with high molecular mass (HMM) Al compounds on CIM disk serum proteins were first separated from LMM-Al species by the use of size exclusion chromatography (SEC). For this purpose 1 mL of serum was injected onto SEC (Superdex 75 HR 10/30) column. Isocratic elution using 0.05 M TRIS-HCl+0.03 M NaHCO(3) was applied and separation of proteins was followed by UV detection at 278 nm. It was experimentally proven that proteins were eluted in 5.5 mL peak that was collected into a polyethylene cup. A 0.1 mL of the sample aliquot was then injected onto the CIM DEAE disk. The separation of serum proteins was obtained in 10 min by applying linear gradient elution from 100% buffer A (0.05 M TRIS-HCl+0.03 M NaHCO(3)) to 100% buffer B (A+1M NH(4)Cl) and followed by UV detection at 278 nm. Separated Al species were detected on-line by inductively coupled plasma mass spectrometry (ICP-MS). Well-resolved protein peaks were obtained. It was experimentally proven that 90+/-3% of Al in spiked serum of renal patient was eluted under the transferrin peak. The proposed speciation procedure removes LMM-Al species and enables reliable determination of the concentration and composition of Al bound to proteins by CIM DEAE-ICP-MS when the concentration of Al in serum is higher than 5 ng mL(-1). In comparison to chromatographic columns CIM disks enable faster separation and simpler manipulation during cleaning procedure and coupling to ICP-MS.