Amino Acids In Liquid Chromatography Research Articles

High-performance liquid chromatography of amino acids, peptides and proteins: CXXI. 8-Hydroxyquinoline-metal chelate chromatographic support: an additional mode of selectivity in immobilized-metal affinity chromatography

In our associated studies, the binding of proteins in immobilized-metal affinity chromatography has been shown to be independent of surface-exposed histidines and aromatic amino acid residues, when hard metals are used. The present investigation documents the behaviour of iminodiacetic acid (IDA) and 8-hydroxyquinoline (8-HQ) immobilized on Sepharose CL-4B and chelated with detergent (Brij-35), at pH 7.0. the 8-HQ gel had a higher capacity for tuna heart cytochrome c (THCC) when Fe 3+ was immobilized than when Al 3+ or Yb 3+ was used, whilst 8-HQ-Cu 2+ and 8-HQ-Ca 2+ did not bind this protein. The equivalent IDA chelates showed no binding of the protein. The THCC was recovered from the 8-HQ-Fe 3+, -YB 3+ and -Al 3+ supports upon elution with high concentrations of phosphate, glutamate or malonic acids, suggesting that acidic amino acid residues were involved in the binding. Application of molecular graphics procedures reveals that the 8-HQ-metals 3+ chelate represents a new class of coordination geometry for binding to proteins, and hence offers an additional mode of selectivity in immobilized-metal affinity chromatographic separations.

High-performance liquid chromatography of amino acids, peptides and proteins CXV. Thermodynamic behaviour of peptides in reversed-phase chromatography

The thermodynamic behaviour of three peptides, bombesin, β-endorphin and glucagon, was studied under reversed-phase high-performance liquid chromatographic conditions. Experimental data related to the interactive surface contact area ( S values) and solute affinity (log k 0) were derived over a range of temperatures between 5 and 85°C. These experimental conditions allowed changes in the secondary structure of the solute to be monitored. The influence of the nature of the stationary phase ligand on the relative conformational stability of the three peptides was analysed by acquiring data with n-octadecyl silica (C 18) and n-butyl silica (C 4) sorbents. Values for the relative changes in entropy and ethalpy associated with the interactive process were also determined. The results provide further insight into the factors involved with the stabilization of secondary structure and the mechanism of the interaction of peptides with hydrophobic surfaces.

High-performance liquid chromatography of amino acids, peptides and proteins : CXIV. Protein interactions with porous coulombic sorbents: comparison of experimental findings with predictions of several adsorption models

The adsorption behaviour of three anion-exchange sorbents, DEAE Trisacryl M, DEAE Sepharose FF and DEAE Fractogel 650 M has been investigated for proteins of different size and charge distribution. The maximum capacity, q m, and the dissociation constant, K d, were found to be protein size dependent. Protein loadings with carbonic anhydrase infer unhindered access to the pores of these three sorbents and thus high capacities. Kinetic profiles, monitoring protein uptake from solution, indicated that the rate of adsorption was fast for small proteins. Mathematical models, derived by others, were adopted to extract values for the interaction rate, k 1, and the effective diffusion, D p. The simplest model of Horstmann et al. [ J. Chromatogr., 361 (1986) 179] gave high values for k 1, confirming that this model lumped together the resistances to mass transfer. Correlation between theory and experiment was best when using the most sophisticated model, that of Arve and Liapis [ AIChE. J., 33 (1987) 179] where the resulting values for k 1 were larger. Diffusion for the smaller proteins agreed well with bulk diffusion, indicating little restriction to mass transfer through the pores. Contrary to this, values of D p for the largest protein, ferritin, were 1 /40th of the free diffusivities, further highlighting the influence of protein size on pore accessibility.

High-performance liquid chromatography of amino acids and depeptides on new ion exchangers of the HEMA series

The separation of a number of essential amino acids and dipeptides was studied in ion-exchange systems based on poly(hydroxyethyl methacrylate) stationary phases modifed with various ionizing groups (carboxymethls, a weak cation exchanger; sulphobutyl, a strong cationa exchanger; diethylaminoethyl, a weak anion exchanger) and on a ethylene dimethacrylate-based hydrophobic column. The ion-exchane materials also exhibited hydrophobic properties, caused by the poly(hydroxyethyl methacrylate) matrix. The dependence of the capacity factros on the mobile phase pH, the ionic strength, the type and concentration of the displacer salt and the content of the organic modifier (methanol) was investigated. The best separations of amino acids and dipeptides, with baseline resolution for many test compounds, was attained with cation exchangers, HEMA Bio 1000 CM and SB. The analytes were detected either by measurement of the absorbance at 210 nm or amperometrically at a copper, at a potential of + 0.15 V (Ag/AgCl) (for separations at pH >6).

High-performance liquid chromatography of amino acids, peptides and proteins: CXI. Retention behaviour of proteins with macroporous tentacle-type anion exchangers

The chromatographic behaviour of various proteins with Fractogel-TMAE and LiChrospher-TMAE tentacle-type strong anion-exchange sorbents has been investigated. In particular, the log {ie versus log {ie (where {ie is the median capacity factor and {ie is the corresponding salt concentration) dependencies for two salt systems (NaCl and KBr) have been documented. The Fractogel-TMAE sorbent exhibited retention characteristics in terms of relative protein charge dependencies (slope Z c) and affinity values (log K c, which is log {ie at c = 10 −6 M, where c is the concentration of the displacer ion) which were similar to the more conventional anion-exchange sorbents such as the Mono-Q sorbent. However, the Z c and log K c values obtained with the LiChrospher-TMAE sorbent were significantly decreased for most proteins. Furthermore, proteins such as carbonic anhydrase and myoglobin were not retained at pH values up to 4 and 2 units above their p I values, respectively. These results illustrate the different adsorptive properties of the tentacle-type sorbents compared to other monolayer or polymer layer ion exchangers in terms of accessibility of the charged ligand and the underlying retention mechanism.

High-performance liquid chromatography of amino acids, peptides and proteins: CXIII. Predicting the performance of non-porous particles in affinity chromatography of proteins

A mathematical model, based on the Langmuir adsorption isotherm, has been developed to predict the performance of bioaffinity chromatography columns, packed with non-porous particles, in protein purification at the laboratory and preparative scales. Both the surface kinetics and the external film resistance as the rate controlling steps have been taken into account. The effects of particle size, fluid flow-rate and adsorbate concentration are examined. The model has been used to determine the forward interaction rate constant ( k 1) for different adsorbate-ligand systems. Examples of the comparison between predicted and experimental breakthrough curves of a lysozyme-Cibacron Blue F3GA biomimetic affinity system are given. The influence of ionic strength and ligand density have also been evaluated.

High-performance liquid chromatography of amino acids, peptides and proteins: CX. Principal component analysis of four sets of group retention coefficients derived from reversed-phase high-performance liquid chromatography of peptides

Principal component analysis procedures have been used to further characteristise peptide retention behaviour in reversed-phase (RP) high-performance liquid chromatography. In particular, the analysis was performed with four new scales of group retention coefficients (GRCs) in conjunction with fourteen physicochemical descriptors of the side chain functionalities of the twenty naturally occurring amino acids. The results demonstrate a negative correlation between GRCs derived from peptide retention data with RP-C18 or RP-C8 stationary phases and aqueous trifluoroacetic acid-acetonitrile solvents and amino acid parameters which describe electronic characteristics. Conversely, there was a positive correlation between GRCs derived from peptide retention data with a RP-C18 stationary phase and aqueous trifluoroacetic acid-acetonitrile-2-propanol mixtures and amino acid parameters related to steric and volume characteristics. These results are interpreted in terms of the solvophobic theory. The relevance of quantitative structure-retention relationships to the mechanistic basis of peptide and protein interactions with chromatographic surfaces is discussed.

High-performance liquid chromatography of amino acids, peptides and proteins: CXII. Analysis of operating parameters affecting the breakthrough curves in fixed-bed chromatography of proteins using several mathematical models

Performance of a packed bed, in terms of adsorbent utilization and kinetic characteristics, was studied for the adsorption of HSA to the ion-exchange resins, DEAE Trisacryl M and DEAE Sepharose FF. The influence of column length, temperature and feed concentration on the shape of the breakthrough curve has been investigated and the resulting operating capacities, q * measured by graphical integration. Mathematical models prevalent in the literature have been used to extract out the kinetic parameters, the effective diffusivity, D p, and the interaction rate constant, k 1, from the experimental breakthrough curves, and these parameters have been used to evaluate the performance of the columns and to validate the models selected. The effect of protein size on the adsorption behaviour was also investigated using the proteins HSA, transferrin and ferritin, determining q *, k 1, and D p from the breakthrough curves.

High-performance liquid chromatography of amino acids, peptides and proteins : CVII. A Analysis of group retention contributions for peptides separated with a range of mobile and stationary phases by reversed-phase high-performance liquid chromatography

An extensive data base which comprises the retention data of a total of 2106 peptides has been established and used to derive individual amino acid group retention coefficients. A multiple linear regression matrix approach was employed for solving the numerical value of the coefficients from the multivariate structure—retention dependencies. Statistical analysis of the retention data revealed that a minimum of 100 peptides is required to provide consistent values of the amino acid coefficients. Categorisation of all peptides allowed the influence of various chromatographic parameters on the coefficients to be evaluated. In particular, a decrease in the alkyl chain length of the chemically modified n-alkylsilica from octadecyl to butyl did not generally coincide with a decrease in the value of the group retention coefficients of individual amino acids. This study has established a detailed computational basis for characterising peptide retention behaviour and provides further insight into the mechanism of the interaction of peptides with immobilised hydrocarbonaceous ligands.

High-performance liquid chromatography of amino acids, peptides and proteins : CIX. Investigations on the relation between the ligand density of Cibacron Blue immobilized porous and non-porous sorbents and protein-binding capacities and association constants

A porous silica of nominal 5 μm particle diameter and 30 nm pore size (nucleosil 300-5) and a non-porous silica of nominal 1.5 μm particle diameter were activated with 3-mercaptopropyltriethoxysilane(MPTS), followed by the immobilization of the triazine dye, Cibacron Blue F3GA. Various Biomimetic dye sorbents with graduated ligand densities betwen 1 μmol/m 2 and 0.01 μmol/m 2 were prepared. The capacities and the association constants associated with the binding of lysozyme to these sorbents were determined by frontal analysis experiments [J. Chromatogr., 476 (1989) 205-225]. Due to the ability of the Cibacron Blue F3GA-modified silicas to act as mixed mode coulombic and hydrophobic interaction sorbents and the highly charged nature of the surface structure of lysozyme (p I 11), two mobile phase conditions were examined. In one case a 0.1 M sodium chloride −0.1 M phosphate buffer, pH 7.8 was employed as the equilibration and loading buffer to monitor the influence of ionic interactions. The elution was performed in each case with a 2.5 M potassium thiocyanate solution. With the porous silica dye sorbents and 1 M NaCl present in the loading buffer, the highest capactiy was achieved when Cibacron Blue F3GA was immobilised to the level of 0.1 μmol/m 2. In the case of the non-porous silica dye sorbents, the maximum protein capacity was achieved when 0.5 μmol/m 2 dye were immobilised onto the support. Evaluation of the frontal breakthrough curves confirmed that the kinetics of adsorption of lysozyme onto the non-porous sorbent were substantially faster than the adsorption of lysozyme onto the porous sorbent due to the absence of pore diffusion effects in case of the non-porous

High-performance liquid chromatography of amino acids, peptides and proteins : C. Characterisation of coulombic interactive regions on hen lysozyme by high-performance liquid anion-exchange chromatography and computer graphic analysis

The molecular characteristics of the dominant anion-exchange binding site of hen egg white lysozyme (HEWL) has been investigated using a combination of high-performance liquid chromatographic techniques and computer graphic analysis of the X-ray crystallographic structure. These studies have indicated that the site of highest electrostatic potential, in terms of the density of negatively charged amino acid side chains, is located around the catalytic cleft area. The four residues tentatively identified to be involved in the electrostatic binding domain were aspartic acid 48, 52, 101 and glutamic acid 35. The number of these charged groups correlated with the maximum value of the chromatographically determined retention parameter (Z c value). Variations in the range of experimental Z c values obtained under different elution conditions have been interpreted in terms of comformational flexibility of the structureal domains of HEWL which result in the opening or closure of the catalytic cleft during the retention process.

Isolation and characterization of hepatotoxic microcystin homologs from the filamentous freshwater cyanobacterium Nostoc sp. strain 152

A strain of the filamentous cyanobacterium Nostoc sp. isolated from a lake in Finland was found to produce at least nine hepatotoxic peptides with chemical and toxicological properties similar to those of the hepatotoxic hepta- and pentapeptides produced by other cyanobacteria. Toxins were isolated and purified by high-performance liquid chromatography. Amounts available for five of the purified toxins (P6, P14, P15, P16, and P18) were adequate for high-performance liquid chromatography amino acid analysis and determination of molecular weight by fast-atom bombardment-mass spectrometry (FAB-MS). Quantities of three toxins (P14, P15, and P16) were adequate for further analysis by high-resolution FAB-MS, FAB-MS/MS, and 1H-nuclear magnetic resonance. Analysis showed that the toxins are new types of microcystin-LR homologs. Microcystin-LR contains equimolar amounts of D-alanine, L-leucine, D-erythro-beta-methylaspartic acid, L-arginine, ADDA (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid), D-glutamic acid, and N-methyldehydroalanine (molecular weight [MW], 994). Nostoc sp. strain 152 was found to produce the following microcystin-LR homologs: (i) P6 contains an extra methylene group most probably due to the presence of N-methyldehydrobutyrine instead of N-methyldehydroalanine (MW, 1,008); (ii) P14 is O-acetyl-O-demethyl ADDA-microcystin-LR (MW, 1,022); (iii) P15 is 3-demethyl-O-acetylADDA-homoarginine-microcystin-LR (MW, 1,036); (iv) P16 is 3-demethyl-O-acetyl-O-demethylADDA-microcystin-LR (MW, 1,008); (v) P18 is 3-demethyl-O-acetyl-O-demethylADDA-homoarginine-microcystin- LR (MW, 1,022). The toxicities of the new microcystin homologs were not significantly different from those of microcystin-LR or demethylmicrocystin-LR.

High-performance liquid chromatography of amino acids, peptides and proteins : CIII. Mass transfer resistances in ion-exchange and dye-affinity chromatography of proteins

Adsorption equilibria and rate kinetics have been investigated for the binding of several proteins, with different molecular geometrics, to several ion-exchange and dye-affinity chromatographic resins with varying pore size and protein accessibilities. The pore geometry was shown to play a significant role in the protein capacity and loadability of both the ion-exchange and dye-affinity resins. For example the Fractogel HW75—Cibacron Blue F3GA affinity sorbent had the greatest capacity for the small protein, lysozyme, compared to the other Fractogel HW-Cibacron Blue F3GA sorbents, and similarly, the ion-exchange resins, such as DEAE-Fractogel 65, bound more human serum albumin (HSA), as opposed to the larger protein, ferritin. The apparent diffusion of protein from the bulk phase to the ligands/ionic sites was calculated to be considerably restricted when the pore to protein size ratio was small, as is the case of DEAE Fractogel 65/ferritin system, and the dye-affinity Fractogel HW55/HSA system. In these circumstances, pore diffusivity was calculated to be up to 100-fold smaller than bulk diffusivity.

High-performance liquid chromatography of amino acids, peptides and proteins : XCIX. Comparative study of the equilibrium refolding of bovine, procine and human growth hormone by size-exclusion chromatography

The equilibrium refolding of bovine, porcine and human growth hormone and ovine prolactin in guanidine hydrochloride has been investigated using high-performance size-exclusion chromatography (HPSEC). It was found that bovine an porcine growth hormones exhibited very similar refolding behaviour. However, the renaturation of human growth hormone followed a different pathway. In particular, the folding transition of human growth hormone occured at 4.7 M guanidine hydrochloride compared to 3.8 and 3.5 M for the bovine and porcine molecules, respectively, and 3.5 M for ovine prolactin. The refolding mechanism of an internally clipped fragment derived from partial tryptic digestion, exhibited similar folding properties to the corresponding intact molecule. The internally clipped analogue existed as a relatively larger molecule under fully denaturing conditions. Reduction followed by carboxymethylation resulted in growth hormone molecules with significantly reduced stability and altered folding properties. The results have been correlated with differences in structure to further demonstrated the utility of HPSEC in the study of protein folding and stability.

High-performance liquid chromatography of amino acids, peptides and proteins : XCVII. The influence of the gradient elution mode and displacer salt type on the retention properties of closely related protein variants separated by high-performance anion-exchange chromatography

The influence of different elution modes, gradient times and flow-rates on the relative retention of closely related variants of carbonic anhydrase and ovalbumin has been investigated using high-performance ion-exchange chromatography. Three isoform species of carbonic anhydrase and four isoforms related to ovalbumin eluted by anion-exchange chromatography were characterised by isoelectric focusing and sodium dodecylsulphate-polyacrylamide electrophoresis. Gradient retention data were collected using several different alkali metal halides as the displacer salt, in order to systematically evaluate the efffect on selectivity of different anions and cations in the series F −, Cl − and Br −, and Li +, Na + and K +. While the selectivity between the different ovalbumin isoform species remained essentially constant with each displacer salt, solute Z c-values [ J. Chromatogr., 458 (1988) 27] varied with the type of salt. In contrast, non-parallel retention plots were obtained for the carbonic anhydrase isoforms with the Z c values different for each isoform. Furthermore, significant differences in chromatographic behaviour for these proteins were observed between experiments carried out under gradient elution conditions with either varied gradient time and constant flow-rates or fixed gradient time and varied flow-rates. These results are discussed in tems of the influence of column residence time and protein-salt interactions of the solute's interactive ionotope and the concomitant effects these structural perturbations may have on chromatographic behaviour.

High-performance liquid chromatography of amino acids, peptides and proteins : CI. Identification and characterisation of coulombic interactive regions on sperm whale myoglobin by high-performance anion-exchange chromatography and computer-graphic analysis

A combination of high-performance liquid chromatographic and computer-graphic analysis of the electrostatic interactive surface of sperm whale myoglobin has allowed the putative anion-exchange binding domains to be identified and characterised. These studies have established the existence of four regions of high electrostatic potential in terms of negativity charged amino acid side chain groups. These binding sites are comprised of seven glutamic acid residues, two aspartic acid residues and two propionic acid groups located on the heme moiety which are arranged in a continuous topographic band around on section of the myoglobin surface. The number of charged groups in these interactive patches correlated with the range of experimentally determined Z c values under different elution conditions of varying gradient time, flow-rate and displacer salt. The changes in Z c values observed under some chromatographic conditions have been interpreted in terms of the conformational reorientation of the myoglobin molecule as it interacts with the charged stationary phase surface.

A comparison of pulsed amperometric detection and conductivity detection of underivatized amino-acids in liquid chromatography

Pulsed amperometric detection (PAD) in tandem with conductivity detection (CD) has been applied to the direct detection of amino-acids by liquid chromatography. Although sensitive, PAD has a limited linear range of response. Sequential use of conductimetric detection and then PAD extends the dynamic range of amino-acid determination.

High-performance liquid chromatography of amino acids, peptides and proteins : XCV. Thermodynamic and kinetic investigations on rigid and soft affinity gels with varying particle and pore sizes: Comparison of thermodynamic parameters and the adsorption behaviour of proteins evaluated from bath and frontal analysis experiments

The thermodynamic constants, associated with the interaction of three proteins with triazine dye affinity sorbents, have been derived from bath and frontal analysis experiments. In cases where mass-transfer restrictions are very high, calculation of the thermodynamic constants directly from frontal analysis experiments could not be achieved. In such cases, a portion of the adsorbate was always present in the effluent, a situation which has its effect as the split peak phenomenon. With Fractogel-based triazine dye affinity sorbents none of the test proteins applied in frontal analysis were adsorbed. A similar behaviour was observed for a Cellufine sorbent during the adsorption of human serum albumin and the Blue Sepharose CL6B sorbent during the adsorption of alcohol dehydrogenase, which displayed much slower apparent adsorption kinetics than observed in the bath experiments. These phenomena were shown to be associated with changes in the gel structure, caused in part by the column packing procedure. Silica-based sorbents performed better in the adsorption of lysozyme in the column mode than soft-gel affinity sorbents, as was evident in the higher capacities and steeper breakthrough curves. At high protein concentrations (feedstock concentration > 0.2 mg/ml) breakthrough curves obtained with small- and large-particle-size sorbents, but of constant pore size, were found to be identical. This finding demonstrates that the use of small-particle-size sorbents ( e.g. particle diameter, d p ⩽ 5 μm) for the preparative isolation of proteins may not be justified when operating in the overload mode. With other higher-molecular-weight proteins and the silica-based sorbent systems examined, the small-particle-size sorbents ( d p = 5 μm) displayed less symmetrical shapes of their breakthrough curves than the larger-particle-size and soft-gel sorbents. This behaviour was further exacerbated when non-porous glass or silica-based sorbents were utilized. These non-porous affinity sorbents displayed nearly rectangular breakthrough shapes at the onset of the adsorption process, but comparatively slow adsorption kinetics became evident as saturation was approached. This phenomenon has been attributed to surface rearrangement and/or reorientation of the adsorbed proteins, particularly with sorbents of high ligand densities.

High Performance Liquid Chromatography of Amino Acids and Peptides on Strong Cation-Exchange Polymer Gel

The separation of amino acids and peptides on a strong cation-exchange polymer column (Polyspher PE A) has been investigated. Retentions of hydrophobic amino acids and peptides were affected by hydrophobic interaction with the polymer matrix, which was useful for separation of peptides with close pi values. Simultaneous separation of acidic, neutral and basic peptides was successfully performed by linear gradient elution.

Precolumn Phenylisothiocyanate Derivatization and Liquid Chromatography of Amino Acids in Food

A precolumn phenylisothiocyanate derivatization method is described for the determination of amino acids in protein hydrolysates from a wide variety of complex food matrixes, with and without performic acid oxidation pretreatment. Analysis of samples that were not pretreated with performic acid was necessary since this pretreatment destroyed an average of 25% of the histidine and 87% of the tyrosine present in the food samples. This method is rapid and reproducible; coefficients of variation between duplicate analyses of the same food item were less than 5% for a majority of the amino acids. Occasionally, variation between duplicate analyses for histidine and tyrosine was greater than 10%. Recoveries of amino acids added to samples were in the 100% range.