E.C. Nice

Hydrophobic high-performance liquid chromatography of hormonal polypeptides and proteins on alkylsilane bonded silica

Thirty-two hormonal polypeptides and nine proteins (8-65 kD) have been used to evaluate the potential of high-performance liquid chromatography on alkylsilane-bonded silica for separating and recovering biologically active compounds of this type. The basic method used was gradient elution with acetonitrile in an acid phosphate buffer. Variation of key chromatographic parameters demonstrated that low pH (less than 4.0) and high buffer molarity (greater than 0.1 M) are mandatory for reproducible high efficiency polypeptide chromatography. Simple NaCl-HCl mixtures of appropriate acidity and molarity could be substituted for the acid phosphate buffer, with the advantage of minimising non-physiological ion contributions to eluted materials. Minor selective effects were noted with different organic modifiers, but variation of other parameters, including choice of specific alkylsilane packings, did not materially influence separations. Under optimal conditions all of the polypeptides tested could be efficiently chromatographed, and many simultaneously resolved, as could most of the proteins tested. Three of the more hydrophobic proteins could not, however, be eluted from the alkylsilane packings. Retention orders of smaller compounds (less than 15 residues) generally correlated with the sum of the Rekker fragmental constants of their strongly hydrophobic residues. Larger polypeptides showed numerous anomalies when ranked by this means, however, limiting its predictive value. The separation of at least eighteen discrete components from a partially-purified posterior pituitary extract has demonstrated the capability of alkylsilane-type reversed-phase packings for the hydrophobic high-performance liquid chromatography of complex biological mixtures.

Instrumental biosensors: new perspectives for the analysis of biomolecular interactions

The use of instrumental biosensors in basic research to measure biomolecular interactions in real time is increasing exponentially. Applications include protein-protein, protein-peptide, DNA-protein, DNA-DNA, and lipid-protein interactions. Such techniques have been applied to, for example, antibody-antigen, receptor-ligand, signal transduction, and nuclear receptor studies. This review outlines the principles of two of the most commonly used instruments and highlights specific operating parameters that will assist in optimising experimental design, data generation, and analysis.

Comparison of short and ultrashort-chain alkylsilane-bonded silicas for the high-performance liquid chromatography of proteins by hydrophobic interaction methods

Abstract The optimization of the reversed-phase high-performance liquid chromatography of proteins has beene xamined using a series of maximum-coverage alkylsilane-bonded silica packings with different carbon chain-lengths (C1–C18). The greatest range of individual compounds that could be successfully chromatographed was obtained with 200 h) of these columns under acid (pH 2.1) conditions. This system has been used to separate prolactin and growth hormone (22 kD) from biological samples by employing a trace-enrichment step on 10-μm tap-packed short alkyl-chain columns prior to gradient-elution chromatography on a 5-μm particle-size (8 nm pore-size) Ultrasphere SAC column.

High-pressure liquid chromatography of steroids secreted by human adrenal and testis cells in monolayer culture

Reversed-phase high-pressure liquid chromatography with gradient elution on Zorbax-ODS columns has been used to separate, identify, and measure, spectrophotometrically, the steroids secreted by both human adrenal and testis cells in primary monolayer culture. Three related systems using exponential concave gradients have been developed with the specific objective of resolving the steroids produced by these two tissues. A methanol-water gradient has been used to separate most adrenal steroids, an acetonitrile-water gradient to separate testis steroids, and a dioxane-water gradient to separate polar steroids, including aldosterone. These three systems together permit the resolution of at least 43 naturally occurring steroids, plus four synthetic steroids with adrenocortical activity, with overall total elution times of 1 h or less for each system. Retention data for these steroids are given and the separation of steroids in the biological samples illustrated.

Factors influencing chromatography of proteins on short alkylsilane-bonded large pore-size silicas☆

Abstract The separation of proteins by high-performance gradient chromatography on short alkylchain bonded silica was studied with respect to pore size, mobile-phase composition, and temperature. Selectivity could be increased in particular cases by varying temperature or eluate compositon. Recovery of late-eluting, hydrophobic proteins was found to increase with flow rate and gradient slope. Recovery was also shown to be dependent on eluate composition—decreasing with added salt. The applicability of reverse-phase chromatography to proteins as large as 150 kD was demonstrated by the separation of monoclonal immunoglobulin.

Effects of chain length and carbon load on the performance of alkyl-bonded silicas for protein separations

Abstract The retention and recovery of proteins in reversed-phase liquid chromatography with gradient elution using aqueous-organic mobile phases were found to be similar from large pore (30 nm) silica bonded to maximal extents with either propylor octyldimethylsilanes. Proteins were less retained and less completely recovered from silica partially bonded with octyldimethylsilane when used with an acetonitrile-containing mobile phase but not with 1-propanol as modifier. The elution position of proteins under gradient conditions was shown to be independent of flow-rate for fixed gradient slope, but to move later in the gradient with increases in gradient slope. The effects of gradient slope and flow-rate on resolution, separation time, and sensitivity are illustrated.

Purification and Characterization of a Novel Restricted Antigen Expressed by Normal and Transformed Human Colonic Epithelium

A cell surface antigen that is expressed by normal and 95% of transformed colonic epithelium and is recognized by the monoclonal antibody A33 (Welt, S., Divgi, C. R., Real, F. X., Yeh, S. D., Garin-Chesa, P., Finstad, C. L., Sakamoto, J., Cohen, A., Sigurdson, E. R., Kemeny, N., Carswell, E. A., Oettgen, H. F., and Old, L. J. (1990) J. Clin. Oncol. 8, 1894-1906) has been purified to homogeneity from the human colonic carcinoma cell line LIM1215. The A33 protein was purified from Triton X-114 extracts of LIM1215 cells under nondenaturing conditions. These extracts were applied sequentially to Green-Sepharose HE-4BD, Mono-Q HR 10/10, Superose 12 HR 10/30, and micropreparative Brownlee Aquapore RP 300. The purification was monitored by biosensor analysis using surface plasmon resonance detection with a F(ab′)2 fragment of the humanized A33 monoclonal antibody immobilized on the sensor surface and Western blot analysis following SDS-polyacrylamide gel electrophoresis (PAGE) under nonreducing conditions using humanized A33 monoclonal antibody. The purified A33 antigen has a Mr on SDS-PAGE of 43,000 under nonreducing conditions. By contrast, the purified protein displayed a Mr of approximately 180,000 under native conditions on both size exclusion chromatography and native PAGE, possibly due to the formation of a homotetramer. N-terminal amino acid sequence analysis of the purified protein identified 34 amino acid residues of a unique sequence: ISVETPQDVLRASQGKSVTLPXTYHTSXXXREGLIQWD. A polyclonal antibody was raised against a synthetic peptide corresponding to residues 2-20 of this sequence. The antipeptide serum recognized the purified protein using Western blot analysis under both nonreducing (Mr 43,000) and reducing (Mr 49,000) conditions.

Murine epidermal growth factor: heterogeneity on high resolution ion-exchange chromatography.

We have shown that epidermal growth factor (EGF) purified either by the classical method of Savage and Cohen, or solely by h.p.l.c. techniques can be resolved into two species, EGF alpha and EGF beta. However, despite the apparent purity of such materials, as determined both chromatographically and by amino acid analysis, they failed to give homogeneous products on radioiodination. Analysis by isoelectric focusing on agarose gels followed by transfer to nitrocellulose and silver staining showed that EGF alpha could be further resolved into three sub‐species which focused at pH 4.6, 4.3 and 4.1. EGF beta (which also focused at pH 4.6) contained very small amounts of the species with isoelectric points of 4.1 and 4.3, probably due to slight contamination of this preparation by EGF alpha. Preparative separation of the sub‐species of EGF alpha was achieved by high performance anion‐exchange chromatography at pH 6.5 on a Pharmacia Mono Q column. Radioiodination of these purified sub‐species did not produce significant charge heterogeneity. However, two slightly different forms of [125I]EGF alpha 1 (pH 4.6 species) were separable by anion‐exchange chromatography on the Mono Q column. All of the EGF species competed for binding to EGF receptors on A431 cells and were active mitogens for BALB/c 3T3 fibroblasts.

Use of hydrophobic interaction methods in the isolation of proteins from endocrine and paraendocrine tissues and cells by high-performance liquid chromatography.

We have recently described the separation of a large number of polypeptide hormones, related peptides and some protein standards by hydrophobic interaction high-performance liquid chromatography (HPLC). This paper reports the practical application of these methods to the reproducible isolation and separation of components of a mixture of immunoreactive calcitonin-like proteins (less than 25 kD) synthesised and secreted by human tumour cells in vitro. Using hydrophobic interaction HPLC on ODS-silica for both preliminary bulk fractionation and subsequent analytical separation greater than 80% recoveries of small (ng) quantities of immunoreactive proteins were obtained from samples containing less than 100 mg total protein, and characteristic profiles of synthesised and secreted materials were established. Using a partially purified hypothalamic extract, containing a number of small proteins (12--25 kD), we have also examined the effects of varying chromatographic conditions in an attempt to modify the separations obtained with ODS-silica using an acid-saline-acetonitrile gradient elution system at ambient temperature, and achieve further resolution of its components. No useful selective effects were observed when temperature, organic modifier, gradient profile or hydrophobic stationary phase were altered. These techniques may not therefore be inherently capable of completely resolving all components of natural protein mixtures. They do, however, offer an adjunct to and in certain cases a substitute for conventional methods of protein separation.

Binding of fluoresceinated epidermal growth factor to A431 cell sub-populations studied using a model-independent analysis of flow cytometric fluorescence data.

A method is developed for determining ligand‐cell association parameters from a model‐free analysis of data obtained with a flow cytometer. The method requires measurement of the average fluorescence per cell as a function of ligand and cell concentration. The analysis is applied to data obtained for the binding of fluoresceinated epidermal growth factor to a human epidermoid carcinoma cell line, A431. The results indicate that the growth factor binds to two classes of sites on A431 cells: 4 X 10(4) sites with a dissociation constant (KD) of less than or equal to 20 pM, and 1.5 X 10(6) sites with a KD of 3.7 nM. A derived plot of the average fluorescence per cell versus the average number of bound ligands per cell is used to construct binding isotherms for four sub‐populations of A431 cells fractionated on the basis of low‐angle light scatter. The four sub‐populations bind the ligand with equal affinity but differ substantially in terms of the number of binding sites per cell. We also use this new analysis to critically evaluate the use of ‘Fluorotrol’ as a calibration standard in flow cytometry.