Hyperplasia in the rabbit bladder urothelium following partial outlet obstruction. Autoradiographic evidence

Recombinant histidine-tagged v-Ha-ras (his-ras) was purified to homogeneity from extracts ofE. coli M15 using a one-step procedure which involved immobilised metal ion chromatography on Ni²⁺-nitriloacetic acid agarose (Ni-NTA). The optimal pH for elution by imidazole was 6.6 and the yield of his-ras protein (greater than 95% pure) was about 4 mg/litreE. coli culture. Chromatography of a mixture of purified his-ras and rat brain cytosol on Ni-NTA together with SDS-PAGE and silver staining of proteins were employed to search forras-binding proteins present in rat brain cytosol. Chromatography of rat brain cytosol alone on Ni-NTA revealed several protein species which were not readily eluted with imidazole. These are likely to be low-abundance brain metal ion binding proteins. Pre-treatment of rat brain cytosol with Ni-NTA before a second round of chromatography on Ni-NTA removed most of these proteins. Chromatography of a mixture of pre-treated rat brain cytosol and purified his-ras protein revealed four new protein bands with molecular weights of 250, 90, 80 and 70 kDa. These were considered to be candidateras-binding proteins. It is concluded that the use of his-ras and immobilised metal ion chromatography does provide an approach which can be used to identifyras binding proteins present in cellular extracts.Abbreviations his-ras histidine-tagged vHa-ras - Ni-NTA Ni²⁺ nitriloacetic acid agarose - IPTG isopropyl thio--D-galactoside     

Purification and Characterization of Antioxidant Peptides from Leukocyte Extract of Crocodylus siamensis

Antioxidant peptides were isolated from the leukocyte extract of the Siamese crocodile, Crocodylus siamensis. Crocodile leukocyte was extracted by a combination of methods including freeze-thawing, acetic acid extraction and homogenization. The peptides in the leukocyte extract were purified by anion exchange chromatography and reversed phase-high performance liquid chromatography. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay was used to evaluate the antioxidant activity of the elution peaks at each purification step. As a result, there were two purified peptides exhibiting strong antioxidant activity in reducing free radicals on DPPH molecules. The amino acid sequences of these peptides were determined by LC-MS/MS as TDVLGLPAK (912.5 Da) and DPNAALPAGPR (1,148.6 Da), and their IC₅₀ values were 153.4 and 95.7 μM, respectively. The results of this study therefore indicate that leukocyte extract of C. siamensis contains peptides with antioxidant activity which could be used as a novel antioxidant.     

Peptide-Induced Morphogenesis in the Nematode-Trapping Fungus Arthrobotrys oligospora

The present investigation shows the ability of peptides to induce capture organ formation in Arthrobotrys oligospora when applied in a synthetic low nutrient medium. Under certain conditions casitone was shown to induce capture organ formation. The active principle in casitone was concentrated and purified by alternating procedures of ion exchange chromatography and gel chromatography in pyridine-acetic acid buffers. Crude casitone solutions were applied to columns of Dowex 50 W-X2 and eluted stepwise with 0.1–1.0 M pyridine-acetic acid pH 3.2–5.1. Active portions, free from most acid and neutral amino acids, were further purified on columns of Sephadex G-10 in 0.1 M pyridine-acetic acid pH 4.6. Aromatic amino acids and large molecules in the void volume could be separated from an active peptide mixture which was subjected to renewed ion exchange chromatography on Bio-Rad AG 50 W-X2. By stepwise and/or gradient elution in 0.1–0.5 M pyridine-acetic acid pH 3.2 fairly purified peptides were obtained. The composition of the test medium is an important factor in spontaneous capture organ formation. The peptides isolated from casitone induced capture organ formation, when given to the fungus in a synthetic mineral salt medium supplied with thiamin and biotin. Similar effects were obtained with small synthetic peptides in the same concentration (0.1 mg/ml). A large variety of peptides seem to be active when applied in a suitable medium. This was especially true for peptides with Rf > Rf_leu on thin layers of cellulose developed with butanol-acetic acid-water (4: 1: 1). Of the peptides investigated valyl-peptides exerted the most drastic effect.     

Optimization of Immobilized Gallium (III) Ion Affinity Chromatography for Selective Binding and Recovery of Phosphopeptides from Protein Digests

Although widely used in proteomics research for the selective enrichment of phosphopeptides from protein digests, immobilized metal-ion affinity chromatography (IMAC) often suffers from low specificity and differential recovery of peptides carrying different numbers of phosphate groups. By systematically evaluating and optimizing different loading, washing, and elution conditions, we have developed an efficient and highly selective procedure for the enrichment of phosphopeptides using a commercially available gallium(III)-IMAC column (PhosphoProfile, Sigma). Phosphopeptide enrichment using the reagents supplied with the column is incomplete and biased toward the recovery and/or detection of smaller, singly phosphorylated peptides. In contrast, elution with base (0.4 M ammonium hydroxide) gives efficient and balanced recovery of both singly and multiply phosphorylated peptides, while loading peptides in a strong acidic solution (1% trifluoracetic acid) further increases selectivity toward phosphopeptides, with minimal carryover of nonphosphorylated peptides. 2,5-Dihydroxybenzoic acid, a matrix commonly used when analyzing phosphopeptides by matrix-assisted laser desorption/ionization mass spectrometry was also evaluated as an additive in loading and eluting solvents. Elution with 50% acetonitrile containing 20 mg/mL dihydroxybenzoic acid and 1% phosphoric acid gave results similar to those obtained using ammonium hydroxide as the eluent, although the latter showed the highest specificity for phosphorylated peptides.     

Isolation of ACTH1-39,ACTH1-38 and CLIP from the calf anterior pituitary

Calf anterior pituitaries were defatted and homogenized and peptides were adsorbed from the homogenate supernatant onto octadecylsilyl-silica. After elution, the resulting extract was subjected to gradient elution reversed-phase high pressure liquid chromatography (RP-HPLC) using aqueous acetonitrile containing 0.1% ($\text{v}\text{v}$) trifluoroacetic acid (TFA). Radioimmunoassay of column fractions for corticotropin (ACTH) revealed three major areas of immunoreactivity. Each was purified to homogeneity by gradient elution RP-HPLC employing aqueous acetonitrile containing either 0.13% heptafluorobutyric acid ($\text{v}\text{v}$) or 0.1% TFA ($\text{v}\text{v}$). Amino acid analysis and exopeptidase and trypsin digestions revealed the three forms of corticotropin to be ACTH_1–38, corticotropin-like intermediary lobe peptide, (CLIP, ACTH_18–39) and ACTH_1–39. ³H-labeled ACTH_1–39 did not give rise to either ³H-ACTH_1–38 or ³H-CLIP during isolation.     

Fluorescent tracer method for protein SH groups: III. Use ofN-(7-dimethylamino-4-methyleoumarinyl) malelmide as a tracer of cysteine-containing peptides

We studied whetherN-(7-dimethylamino-4-methyleoumarinyl) maleimide (DACM) could be used as a fluorescent tracer for the purification and analysis of cysteine-containing peptides. An addition product of DACM with SH compound was stable at room temperature in the solution of pH 9.0 and in 50% acetic acid. However, it was hydrolyzed when heated at 110°C for 48 h in 6n HCl. On filter paper, 1 pmol of the addition product in a spot 3 mm in diameter was visible under ultraviolet illumination. The addition product was also stable on filter paper for at least several months after spotting. The elution velocity of the addition products with low molecular weight SH compounds in the Sephadex G-25 column was low considering their molecular weights. However, in general, the elution velocity increased with an increase in the molecular weight of the addition product. The addition product with a peptide having cysteinyl residue at an N-terminal showed another abnormally retarded peak in elution profile which presumably corresponded to a cyclic compound, a thiazane derivative. However, it was shown on the C-terminal tryptic peptide of actin (Cys Phe) that the conversion to a thiazane derivative could be avoided by hydrolyzing the succinimide portion of DACM at pH 9.0 before digestion. The R_f values on paper chromatography for the addition products also depended on their molecular weights. However, the hydrophobicity of the coumarin portion of DACM and of the side chain of amino acid residues also affected the value. It was concluded that DACM was a valuable reagent for the purification and analysis of cysteine-containing peptides.     

Immobilized metal affinity chromatography using open tubular capillary for phosphoprotein analysis: comparison between polymer brush coating and surface functionalization

In the present article, open tubular-IMAC columns, functionalized by iminodiacetic acid (IDA) for the immobilization of Fe(3+), were prepared by in situ chemical modification of fused silica capillary using two chemistries, polymer brush coating and surface functionalization. One column was based on a poly-(glycidyl methacrylate) brush (GMA) and the other on 3-glycidoxypropyltrimethoxysilane (GLYMO). Phosphopeptide enrichment on the open tubular columns was evaluated on an α(S1), α(S2) mixture and β casein peptides. The optimized enrichment protocol includes sample loading in a slightly acidic solution made with pure deionized water, a washing step with 10% acetonitrile, 0.1% formic acid, and an elution step with 50% acetonitrile, 0.1% phosphoric acid at pH 8.0. MALDI-TOF spectra generated from eluted fractions show several phosphorylated peptides. For example, 7 phosphorylated peptides of the α(S1), α(S2) casein mixture were identified, including a pentaphosphorylated peptide. In terms of selectivity, the two proposed chemistries exhibit different behaviors: the GMA-IDA-Fe(3+) IMAC polymer brush column elutes all phosphorylated peptides in one fraction independently of phosphorylation degree, whereas the GLYMO-IMAC polymer brush provides longer elution times for higher phosphorylation states. In particular, the pentaphosphorylated peptide was eluted after a 30 min elution versus 5 min for monophosphorylated species (isocratic gradient).     

DC-based vaccine loaded with acid-eluted peptides in acute myeloid leukemia: the importance of choosing the best elution method

Tumor-associated peptides isolated by acid elution are frequently used for therapeutic immunization against various tumors both in mice and in humans. In acute myeloid leukemia (AML), the frequent accessibility of a large tumor burden allows for extraction of peptides from leukemia cells by using either citrate–phosphate (CP) or trifluoroacetic acid (TFA) buffer. To develop an optimal immunotherapeutic protocol for AML patients, we evaluated both in mice and in humans, the immunogenicity of peptides eluted from leukemia cells with the two acids (TFA or CP). Although ex vivo studies in mice showed that both prophylactic immunizations with mature dendritic cells (DC) loaded with TFA-peptides (DC/TFA), or CP-peptides (DC/CP), were able to stimulate specific antileukemia immune responses, only vaccination with DC/TFA was able to prevent leukemia outgrowth. Moreover, in humans, only DC/TFA generated significant antileukemia CD4⁺ and cytotoxic CD8⁺ T cell responses in vitro. In summary, these data demonstrate that the choice of the acid elution procedure to isolate immunogenic peptides strongly influences the efficacy of the antileukemia immune responses. These finding raise essential considerations for the development of immunotherapeutic protocols for cancer patients. In our model, our results argue for the use of the TFA elution method to extract immunogenic AML-associated peptides.Authors of submitted papers declare no conflict of interest or financial interest in the product or in potentially competing products held by them, their spouses and/or minor children.     

Development of metal affinity-immobilized liposome chromatography and its basic characteristics

Metal affinity-immobilized liposome chromatography (MA-ILC) was newly developed as a chromatographic technique to separate and analyze peptides. The MA-ILC matrix gel was first prepared by immobilizing liposomes modified with functional ligands. The functional ligand used to adsorb metal ions was N-hexadecyl iminodiacetic acid (HIDA), which is obtained by attaching a long alkyl chain to an iminodiacetic acid (IDA). Cu(II) ion was first adsorbed on the gel matrix through its complex formation with the HIDA on the surface of the immobilized liposome. Synthetic peptides of various types ranging in size from 5 to 40 residues were then used, and their retention properties on the MA-ILC were evaluated. The retention property of peptides on the MA-ILC by using a usual imidazole elution was compared with the retention property in the case of the immobilized metal affinity chromatography (IMAC) and an immobilized liposome chromatography (ILC). It was found that the retention property of peptides on the MA-ILC has the features of both the IMAC and the ILC; the retention ability of peptides depends on both the number of histidine residues in peptides and the liposome membrane affinity of the peptides. Histidine and tryptophan residues among amino acid residues in peptides indicated a high contribution coefficient for the peptide retention on the MA-ILC, probably due to their metal ion and membrane interaction properties, respectively.     

Comparative specificity of microbial acid proteinases for synthetic peptides. 3. Relationship with their trypsinogen activating ability

The specificities of acid proteinases from Aspergillus niger, Aspergillus saitoi, Rhizopus chinensis, Mucor miehei, Rhodotorula glutinis, and Cladosporium sp., and that of swine pepsin, were determined and compared with ability of the enzymes to activate trypsinogen. Various oligopeptides containing l-lysine, Z-Lys-X-Ala, Z-Lys-(Ala)_m, Z-Lys-Leu-(Ala)₂, and Z-(Ala)_n-Lys-(Ala)₃ (X = various amino acid residues, m = 1–4, n = 1–2) were used as substrates. Of the enzymes which are able to activate trypsinogen, most split these peptides at the peptide bond formed by the carbonyl group of l-lysine. For the peptides to be susceptible to the enzymes it was essential that the chain extended for two or three amino acid residues on the C-terminal side of the catalytic point, and that a bulky or hydrophobic amino acid residue formed the imino-side of the splitting point. The rate of hydrolysis was markedly accelerated by elongation of the peptide chain with l-alanine on the N-terminal side of the catalytic point. Thus, of the substrates used, Z-(Ala)₂-Lys-(Ala)₃ was the most susceptible to the microbial acid proteinases possessing trypsinogen activating ability. On the other hand, M. miehei enzyme and pepsin, which do not activate trypsinogen, showed very little peptidase activity on the peptides.     

Evaluation of the interaction of peptides with Cu(II), Ni(II), and Zn(II) by high-performance immobilized metal ion affinity chromatography

High-performance immobilized metal ion affinity chromatography was utilized to evaluate the adsorption properties of 67 synthetic, biologically active, peptides ranging in size from 5 to 42 residues. The metal ions, Cu(II), Ni(II) and Zn(II), were immobilized by iminodiacetic acid (IDA) coupled to TSK gel 5PW (10 microns). Two types of gradient elution (imidazole and pH) were used to evaluate peptide retention by the metal ions. A decreasing pH gradient and an increasing imidazole gradient eluted the peptides in similar order. IDA-Cu(II) and IDA-Zn(II) showed very similar selectivities for the peptides analyzed; however, IDA-Zn(II) displayed a weaker affinity for the peptides. IDA-Ni(II) showed a slightly different pattern of selectivity. Peptide adsorption effects contributed by the metal-free gel matrix were found to be relatively minor. The concentration and type of salt included in the mobile phase could affect the relative affinities of the peptides for the immobilized metal ions. Retention coefficients were assigned to individual amino acid residues by multiple linear regression analysis. Histidine showed the largest positive correlation with retention, followed by aromatic amino acid residues. Modified N-terminal residues resulted in negative contributions to retention. Analyses of peptide amino acid composition alone allowed prediction of peptide retention behavior on immobilized metal ion affinity columns.     

Selective Isolation of Glycoproteins and Glycopeptides for MALDI-TOF MS Detection Supported by Magnetic Particles

Glycosylation is the most common form of posttranslational modification of proteins (50–80%). The isolation, discovery, and subsequent identification of glycosylated peptides and proteins is becoming more and more important in glycoproteomics and diagnosis. MALDI-TOF mass spectrometry is an ideal technique for identifying peptides and proteins and their corresponding modifications. The enrichment of glycosylated peptides and proteins from different sources can be attained by affinity chromatography supported by functionalized magnetic particles. Covalent coating of magnetic beads with Concanavalin A (ConA) and diboronic acid was performed by carbodiimide and poly-glutaraldehyde methods, respectively. The functionalized beads were employed to establish and optimize protocols for the binding and detection of glycosylated peptides and proteins with respect to an automated workflow and the subsequent detection and identification by MALDI-TOF mass spectrometry. For several model proteins, the capture and identification could be demonstrated by SDS-PAGE and MALDI-TOF mass spectrometry. According to the type of glycosylation (high man-nose, hybrid, or complex type) the different proteins were enriched by ConA or boronic acid–functionalized beads.     

Recombinant Expression and In Vitro Characterisation of Active Huwentoxin-IV

Huwentoxin-IV (HwTx-IV) is a 35-residue neurotoxin peptide with potential application as a novel analgesic. It is a member of the inhibitory cystine knot (ICK) peptide family, characterised by a compact globular structure maintained by three intramolecular disulfide bonds. Here we describe a novel strategy for producing non-tagged, fully folded ICK-toxin in a bacterial system. HwTx-IV was expressed as a cleavable fusion to small ubiquitin-related modifier (SUMO) in the cytoplasm of the SHuffle T7 Express lysY Escherichia coli strain, which allows cytosolic disulfide bond formation. Purification by IMAC with selective elution of monomeric SUMO fusion followed by proteolytic cleavage and polishing chromatographic steps yielded pure homogeneous toxin. Recombinant HwTx-IV is produced with a C-terminal acid, whereas the native peptide is C-terminally amidated. HwTx-IV(acid) inhibited Na_v1.7 in a dose dependent manner (IC₅₀ = 463-727 nM). In comparison to HwTx-IV(amide) (IC₅₀ = 11 ± 3 nM), the carboxylate was ~50 fold less potent on Na_v1.7, which highlights the impact of the C-terminus. As the amide bond of an additional amino acid may mimic the carboxamide, we expressed the glycine-extended analogue HwTx-IV^G36(acid) in the SUMO/SHuffle system. The peptide was approximately three fold more potent on Na_v1.7 in comparison to HwTx-IV(acid) (IC₅₀ = 190 nM). In conclusion, we have established a novel system for expression and purification of fully folded and active HwTx-IV(acid) in bacteria, which could be applicable to other structurally complex and cysteine rich peptides. Furthermore, we discovered that glycine extension of HwTx-IV(acid) restores some of the potency of the native carboxamide. This finding may also apply to other C-terminally amidated peptides produced recombinantly.     

Synthesis of anticancer heptapeptides containing a unique lipophilic β2,2‐amino acid building block

We report a series of synthetic anticancer heptapeptides (H‐KKWβ^2,2WKK‐NH₂) containing eight different central lipophilic β^2,2‐amino acid building blocks, which have demonstrated high efficiency when used as scaffolds in small cationic antimicrobial peptides and peptidomimetics. The most potent peptides in the present study had IC₅₀ values of 9–23 µm against human Burkitt's lymphoma and murine B‐cell lymphoma and were all nonhaemolytic (EC₅₀ > 200 µm ). The most promising peptide 10e also demonstrated low toxicity against human embryonic lung fibroblast cells and peripheral blood mononuclear cells and exceptional proteolytic stability. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Trifluoroethanol‐containing RP‐HPLC mobile phases for the separation of transmembrane peptides human glycophorin‐A,integrin alpha‐1, and p24: analysis and prevention of potential side reactions due to formic acid

Reversed‐phase high‐pressure liquid chromatography analysis and purification of three hydrophobic, aggregation‐prone peptides, composed mainly of the transmembrane (TM) sequence, were performed using elution systems containing 2,2,2‐trifluoroethanol (TFE). The addition of 10–16% TFE to a common mobile phase, such as a water/acetonitrile/propanol (PrOH) or a water/PrOH/formic acid system, markedly improved the chromatographic separation of these peptides. The superior performance of TFE‐containing systems in separating peptides over water/PrOH/formic acid systems [Bollhagen R. et al., J. Chromatogr. A, 1995; 711 : 181–186.] clearly demonstrated that adding TFE to the mobile phase is one of best methods for TM‐peptide purification. Characterization of the potential side reactions using MALDI and ESI‐LIT/Orbitrap mass spectrometry indicated that prolonged incubation of peptides in a mixture of TFE–formic acid possibly induces O‐formylation of the Ser residue and N‐formylation of the N‐terminus of peptides. The conditions for selective removal of the formyl groups from TM peptides were also screened. We believe that these results will expand our ability to analyze and prepare hydrophobic, aggregation‐prone TM peptides and proteins. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

A method for the preparation of adjuvant peptide mimetics of GMDP with the use of monoclonal antibodies and combinatorial libraries of peptides in the format of phage display

A method for the preparation of peptide mimetics of GMDP which could exhibit adjuvant activity without the negative effects of GMDP is described. The search for peptides with GMDP-like adjuvant activity was performed using highly specific monoclonal antibodies against GMDP and combinatorial peptide libraries in the format of phage display. Various elution methods were used for the immunoaffinity enrichment of the libraries during the course of the preparation of highly active and specific peptides. A sole peptide Arg-Val-Pro-Pro-Arg-Tyr-His-Ala-Lys-Ile-Ser-Pro-Met-Val-Asn (RN) was obtained by the elution of phage particles from the immunosorbent with a 1 μM solution of the natural ligand (GMDP). Elution with a buffer with a low pH value (0.1 M glycine-HCl, pH 2.2) gave two other peptides: Ser-Gly-Arg-Val-Ala-Val-Ser-Pro-Asp-Ser-Pro-Leu-Phe-Tyr-Pro (SP) and Arg-Tyr-Gly-Gly-Ser-Val-Leu-Asn-Ile-Glu-Cys-Gln-Phe-Tyr-Gly (RG). Affinity constants for the RN and SP peptides proved to be 3.6 × 10⁸ and 3.5 × 10⁸ M⁻¹, respectively. The specificity of the interaction with the monoclonal antibodies was checked by the competitive displacement of the peptides from the antigen-antibody complex by GMDP. The RN peptide exhibited adjuvant activity similar to that of GMDP, but had no pyrogenic effect characteristic of GMDP. The described method could be used for the search for mimetics of biologically active low-molecular compounds.     

Application of matrix solid-phase dispersion methodology to the extraction of endogenous peptides from porcine hypothalamus samples for MS and LC-MS analysis

In this study, we investigated a novel application of matrix solid-phase dispersion (MSPD) methodology for the extraction of endogenous peptides from porcine hypothalamus tissue samples. Several experimental factors of the MSPD procedure were examined. Finally, silica-based octadecyl was chosen as dispersing material and blended with 0.25 g porcine hypothalamus at a ratio of 5, and 10 mL of 60% acetonitrile with 0.2% formic acid in water was chosen as the extraction and elution solvent. This MSPD extraction method was compared to the classic acid extraction method. More peaks were observed in the MSPD extracts (74±5) by MALDI-TOF MS than in acid extracts (34±5). Moreover, 14 potential endogenous peptides were identified in the MSPD extracts after nanoLC-MS/MS analysis, while only 2 endogenous peptides in the acid extracts. These results indicated that MSPD could be employed as a simple and efficient method for the extraction of endogenous peptides from tissues.     

Theoretical analysis of chromatographic peak asymmetry and sharpness by the moment method using two peptides

The analyses of peak shapes in chromatography are useful in operating chromatographic system. The asymmetry and sharpness of a chromatographic peak are estimated by the reversed-phase adsorption of two standard peptides (angiotensin II bradykinin) on C₁₈. In this work, the average particle diameters of C₁₈ were 5 and 15 μm, while the pore sizes were 100 and 300 Å. The composition of the mobile phase was 50/50 vol. % of a binary mixture of acetonitrile and water with 0.1% TFA, and the particles were packed in a stainless column (4.6×150 mm). The third and the fourth central movement were calculated from the chromatographic elution curves by moment analysis. The peak asymmetry was determined by two theoretical calculations: the asymmetry factor by elution peak analysis and skewness with moment analysis. The sharpness was estimated by the fourth central moment. In this work, the most acceptable skewness was calculated when the pore size was 300 Å. The large excess was observed on small pore size.     

Identification and isolation from breast milk of ceruloplasmin-lactoferrin complex

The presence of a complex of the copper-containing protein ceruloplasmin (Cp) with lactoferrin (Lf) in breast milk (BM) is shown for the first time. In SDS-free polyacrylamide gel electrophoresis (PAGE), electrophoretic mobility of Cp in BM is lower than that of plasma Cp, coinciding with the mobility of the complex obtained upon mixing purified Cp and Lf. Affinity chromatography of delipidated BM on Cp-Sepharose resulted in retention of Lf. SDS-PAGE of the 0.3 M NaCl eluate revealed a single band with M_r ∼ 78,000 that has the N-terminal amino acid sequence of Lf and reacts with antibodies to that protein. Synthetic peptides R-R-R-R (the N-terminal amino acid stretch 2–5 in Lf) and K-R-Y-K-Q-R-V-K-N-K (the C-terminal stretch 29–38 in PACAP 38) caused efficient elution of Lf from Cp-Sepharose. Cp-Lf complex from delipidated BM is not retained on the resins used for isolation of Cp (AE-agarose) and of Lf (CM-Sephadex). Anionic peptides from Cp-(586–597), (721–734), and (905–914)—provide an efficient elution of Cp from AE-agarose, but do not cause dissociation of Cp-Lf complex. When anti-Lf is added to BM flowed through CM-Sephadex, Cp co-precipitates with Lf. Cp-Lf complex can be isolated from BM by chromatography on CM-Sephadex, ethanol precipitation, and affinity chromatography on AE-agarose, yielding 98% pure complex. The resulting complex Cp-Lf(1: 1) was separated into components by chromatography on heparin-Sepharose. Limited tryptic hydrolysis of Cp obtained from BM and from blood plasma revealed identical proteolytic fragments. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 2, pp. 208–215.     

Improved performance of column chromatography by arginine: dye-affinity chromatography

Arginine has been effectively used in various column chromatographies for improving recovery and resolution, and suppressing aggregation. Here, we have tested the effectiveness of arginine as an eluent in dye-affinity column chromatography using Blue-Sepharose, which binds enzymes requiring adenyl-containing cofactors (e.g., NAD). A common eluent, NaCl, showed a broad elution peak with low recovery of lactate dehydrogenase, at most approximately 60% using 2M salt. The recovery decreased as the NaCl concentration was either decreased or increased; i.e., the recovery was maximum at 2M. On the contrary, addition of arginine to the eluent resulted in more than 80% recovery above 0.5M and the recovery was nearly independent of the arginine concentration. The elution peak was much sharper with arginine, leading to elution of more concentrated protein solution. Successful elution of proteins bound to the ATP-agarose resins by arginine was also described.