Analysis of protein-protein interaction by simulation of small-zone size exclusion chromatography. Stochastic formulation of kinetic rate contributions to observed high-performance liquid chromatography elution characteristics.

High-performance liquid chromatography (HPLC) procedures provide size-exclusion chromatography with sufficient speed that the elution characteristics of mixtures of interacting macromolecules are potentially determined by the kinetics of association and dissociation. However, few studies have yet addressed the consequences of interaction kinetics on HPLC analyses or evaluated the potential application of HPLC methods for the qualitative and quantitative interpretation of macromolecular interaction kinetics. An earlier simulation of small-zone chromatography of interacting molecules (Stevens, F. J. 1986. Biochemistry. 25:981-993) has been modified to incorporate the effects of association/dissociation kinetics on elution behavior. The previous assumption of instantaneous equilibration has been replaced by explicit calculation of partial relaxation of complexed and free constituent mixtures during each iteration of the simulation. In addition, a stochastically based formulation has been introduced to determine a velocity probability distribution that emulates the partial intermixing of free and complexed pools during the iteration cycle. The simulation generates bimodal elution profiles representing stable complexed and free components of mixtures for which interaction is characterized by slow kinetics relative to chromatography run times. For mixtures with rapid kinetics, a single-asymmetric peak results. When tested with a large-zone sample such that a plateau of stable concentration is generated, the simulation reproduces previous characterizations based on evaluations of solute continuity equations. Therefore, HPLC may, in many cases be an appropriate basis for techniques by which to evaluate kinetic and affinity characteristics of interacting biomolecules.     

Nonprolyl cis peptide bonds in unfolded proteins cause complex folding kinetics

Folding of tendamistat, an inhibitor of alpha-amylase, is a fast two-state process accompanied by two minor slow reactions, which were assigned to prolyl isomerization. In a proline-free variant, 5% of the molecules still fold slowly with a rate constant of 2.5 s(-1). This reaction is caused by a slow equilibrium between two populations of unfolded molecules. The time constant for this equilibration process, its sensitivity to LiCl and its temperature dependence identify it as a cis-trans isomerization of nonprolyl peptide bonds. Although nonprolyl peptide bonds have the cis conformation populating only approximately 0.15% in unfolded proteins, their large number generates a significant fraction of slow-folding molecules. This emphasizes that heterogeneous populations in an unfolded protein can induce complex folding kinetics on various time scales.     

Pulse response in an immobilized-enzyme column: Theoretical method for predicting elution curves

A theoretical method for predicting the elution profile of a pulse response in an immobilized-enzyme column is proposed. The method is based on a mass balance model, which is extensively used in gel chromatography. To test the method, a pulse of sucrose solution was applied to a column of spherical acrylamide gel in which was entrapped invertase from yeast, and it was eluted with 0.05M acetate buffer at pH 5.0. The elution curves of the substrate and the product were in fairly good agreement with the theoretically calculated ones. The method was extended to the system of reversible as well consecutive reactions.     

Kinetic mechanism and catalysis of a native-state prolyl isomerization reaction

Folding of tendamistat is a rapid two-state process for the majority of the unfolded molecules. In fluorescence-monitored refolding kinetics about 8% of the unfolded molecules fold slowly (lambda=0.083s(-1)), limited by peptidyl-prolyl cis-trans isomerization. This is significantly less than expected from the presence of three trans prolyl-peptide bonds in the native state. In interrupted refolding experiments we detected an additional very slow folding reaction (lambda=0.008s(-1) at pH 2) with an amplitude of about 12%. This reaction is caused by the interconversion of a highly structured intermediate to native tendamistat. The intermediate has essentially native spectroscopic properties and about 2% of it remain populated in equilibrium after folding is complete. Catalysis by human cyclophilin 18 identifies this very slow reaction as a prolyl isomerization reaction. This shows that prolyl-isomerases are able to efficiently catalyze native state isomerization reactions, which allows them to influence biologically important regulatory conformational transitions. Folding kinetics of the proline variants P7A, P9A, P50A and P7A/P9A show that the very slow reaction is due to isomerization of the Glu6-Pro7 and Ala8-Pro9 peptide bonds, which are located in a region that makes strong backbone and side-chain interactions to both beta-sheets. In the P50A variant the very slow isomerization reaction is still present but native state heterogeneity is not observed any more, indicating a long-range destabilizing effect on the alternative native state relative to N. These results enable us to include all prolyl and non-prolyl peptide bond isomerization reactions in the folding mechanism of tendamistat and to characterize the kinetic mechanism and the energetics of a native-state prolyl isomerization reaction.     

A novel method to measure self-association of small amphipathic molecules: temperature profiling in reversed-phase chromatography

Biophysical techniques such as size-exclusion chromatography, sedimentation equilibrium analytical ultracentrifugation, and non-denaturing gel electrophoresis are the classical methods for determining the self-association of molecules into dimers, trimers, or other higher order species. However, these techniques usually require high (mg/ml) loading concentrations to detect self-association and also possess a lower size limit that is dependent on the ability of the technique to resolve monomeric from higher order species. Here we describe a novel, sensitive method with no upper or lower molecular size limits that indicates self-association of molecules driven together by the hydrophobic effect under aqueous conditions. "Temperature profiling in reversed-phase chromatography" analyzes the retention behavior of a sample over the temperature range of 5-80 degrees C during gradient elution reversed-phase high-performance liquid chromatography. Because this technique greatly increases the effective concentration of analyte upon adsorption to the column, it is extremely sensitive, requiring very small sample quantities (microgram or less). In contrast, the classical techniques mentioned above decrease the effective analyte concentration during analysis, decreasing sensitivity by requiring larger amounts of analyte to detect molecular self-association. We demonstrate the utility of this technique with 14-residue cyclic and linear cationic peptides (<2000 Da) based on the sequence of the de novo-designed cytolytic peptide, GS14. The only requirements for the analyte molecule when using this technique are its ability to be retained on the reversed-phase column and to be subsequently removed from the column during gradient elution.     

Identification of diadenosine triphosphate in Brugia malayi by reverse phase high performance liquid chromatography and MALDI mass spectrometry

The presence of diadenosine oligophosphates (ApnA) in eukaryotic pathogens has been difficult technically to assess and thus is often overlooked. ApnA are a family of intercellular and intracellular signaling molecules and their biological activities differ relative to the number of phosphate moieties. The application of mass spectrometry to differentiate nucleotide phosphates has been limited by the high salt content in tissue extracts, enzymatic reactions or high performance liquid chromatography (HPLC) buffers, as well as the potential for sample loss when processing and desalting small biological samples. To address this problem a simple reverse phase HPLC (RP-HPLC) method using volatile organic buffers at low pH was developed to create elution profiles of adenosine and diadenosine phosphates. To test this method on a eukaryotic pathogen, small intravascular human filarial parasites (Brugia malayi) were extracted in phosphate buffered saline and a nucleotide phosphate profile was visualized by RP-HPLC. A major peak eluting at 10.4 min was analyzed directly by mass spectrometry and this confirmed the presence of significant quantities of diadenosine triphosphate, Ap3A. Application of this simplified RP-HPLC method will facilitate research on the normal and pathophysiological effects of ApnA particularly in situations when analysis of small biological samples is required.     

Folding of ribonuclease T1. 1. Existence of multiple unfolded states created by proline isomerization

It is our aim to elucidate molecular aspects of the mechanism of protein folding. We use ribonuclease T1 as a model protein, because it is a small single-domain protein with a well-defined secondary and tertiary structure, which is stable in the presence and absence of disulfide bonds. Also, an efficient mutagenesis system is available to produce protein molecules with defined sequence variations. Here we present a preliminary characterization of the folding kinetics of ribonuclease T1. Its unfolding and refolding reactions are reversible, which is shown by the quantitative recovery of the catalytic activity after an unfolding/refolding cycle. Refolding is a complex process, where native protein is formed on three distinguishable pathways. There are 3.5% fast-folding molecules, which refold within the millisecond time range, and 96.5% slow-folding species, which regain the native state in the time range of minutes to hours. These slow-folding molecules give rise to two major, parallel refolding reactions. The mixture of fast- and slow-folding molecules is produced slowly after unfolding by chain equilibration reactions that show properties of proline isomerization. We conclude that part of the kinetic complexity of RNase T1 folding can be explained on the basis of the proline model for protein folding. This is supported by the finding that the slow refolding reactions of this protein are accelerated in the presence of the enzyme prolyl isomerase. However, several properties of ribonuclease T1 refolding, such as the dependence of the relative amplitudes on the probes, used to follow folding, are not readily explained by a simple proline model.     

Purification of fatty acid ethyl esters by solid-phase extraction and high-performance liquid chromatography

We have developed a two-step method to purify fatty acid ethyl esters (FAEE) using solid-phase extraction (SPE), with a recovery of 70±3% (mean±S.E.M.) as assessed using ethyl oleate as a recovery marker from a standard lipid mixture in hexane. The first step of the SPE procedure involves application of a lipid mixture to an aminopropyl-silica column with simultaneous elution of FAEE and cholesteryl esters from the column with hexane. Gas chromatographic analysis of FAEE without interference from cholesteryl esters may be performed using the eluate from the aminopropyl-silica column, thus eliminating the need for an octadecylsily (ODS) column in this case. The FAEE can then be separated from the cholesteryl esters, if necessary, by chromatography on an ODS column and elution with isopropanol-water (5:1, v/v). Both the aminopropyl-silica and ODS columns were found to be effective for up to four uses. To permit isolation of specific FAEE species following isolation of total FAEE by the two-step SPE method, we have also developed a purification scheme for individaal FAEE by high-performance liquid chromatography (HPLC). Thus, this simple method allows for reproducible isolation of total FAEE by SPE and isolation of individual FAEE species by HPLC.     

Folding and association of the antibody domain CH3: prolyl isomerization preceeds dimerization.

The simplest naturally occurring model system for studying immunoglobulin folding and assembly is the non-covalent homodimer formed by the C-terminal domains (CH3) of the heavy chains of IgG. Here, we describe the structure of recombinant CH3 dimer as determined by X-ray crystallography and an analysis of the folding pathway of this protein.Under conditions where prolyl isomerization does not contribute to the folding kinetics, formation of the beta-sandwich structure is the rate-limiting step. beta-Sheet formation of CH3 is a slow process, even compared to other antibody domains, while the subsequent association of the folded monomers is fast. After long-time denaturation, the majority of the unfolded CH3 molecules reaches the native state in two serial reactions, involving the re-isomerization of the Pro35-peptide bond to the cis configuration. The species with the wrong isomer accumulate as a monomeric intermediate. Importantly, the isomerization to the correct cis configuration is the prerequisite for dimerization of the CH3 domain. In contrast, in the Fab fragment of the same antibody, prolyl isomerization occurs after dimerization demonstrating that within one protein, comprised of highly homologous domains, both the kinetics of beta-sandwich formation and the stage at which prolyl isomerization occurs during the folding process can be completely different.     

Theoretical and experimental studies on zone-interference chromatography as a new method for determining macromolecular kinetic constants

Zone-interference chromatography is a new method for studying macromolecular interactions (S. Endo and A. Wada, Anal. Biochem. 124 (1982) 372). This method is a new style of affinity chromatography which requires no preparation of affinity-column materials but utilizes the velocity difference in a column between interacting molecular species. Using the stochastic theory on the behavior of solute molecules, both the association and the dissociation rate constants can be analytically obtained from the degree of deformation of elution patterns, i.e., the change of the first and second moments. In order to verify the present theory, computer simulation of elution profiles by the extended plate theory and a binding experiment between glutamate dehydrogenase and ADP have been carried out.     

Folding mechanism of porcine ribonuclease

The kinetics of unfolding and refolding of porcine ribonuclease were investigated. The unfolded state of this protein was found to consist of a fast-refolding species (UF) and two slow-refolding species (UIS and UIIS). After the rapid collapse of the structure during the N (native)----UF unfolding reaction, UIS and UIIS are produced from UF by two independent slow isomerizations of the unfolded polypeptide chain, leading ultimately to a mixture of about 10% UF, 20% UIIS and 70% UIS molecules at equilibrium. This is at variance with all other ribonucleases investigated to date, which show a distribution of 20% UF, 60 to 70% UIIS and only 10 to 20% UIS. The two isomerizations of the unfolded porcine protein differ strongly in rate. The first process with tau = 250 seconds (10 degrees C) leads to an increase in the fluorescence of Tyr92 and was identified as cis in equilibrium trans isomerization of Pro93. At equilibrium, most unfolded molecules contain an incorrect trans Pro93. The second isomerization is much slower (tau = 1300 s at 10 degrees C) and leads to a predominance of the incorrect isomer as well. Like isomerization of Pro93, it is governed by an activation enthalpy of 22 kcal/mol (92 kJ/mol) and it was tentatively assigned to the Pro114-Pro115 sequence of porcine ribonuclease. Because of the wide separation in rate between the two reactions, molecules with an incorrect isomer only at Pro93 accumulate transiently after unfolding. These are the UIIS molecules. Most of them are finally converted to UIS by the 1300 second process. All molecules that have undergone this isomerization refold very slowly, i.e. the UIS molecules. The major fraction contains two incorrect isomers. A 1300 second isomerization after unfolding and a predominant very slow refolding reaction were observed only for the porcine protein. We suggest that these changes in the folding mechanism may be correlated with the presence of the Pro114-Pro115 sequence, which occurs only in porcine ribonuclease. The refolding pathway of porcine UIIS involves the rapid formation of a native-like intermediate with an incorrect trans Pro93 as was found previously for the bovine ribonuclease, where the UIIS species predominates in the unfolded state.     

An electrophoretic method to deliver topical drugs to the eye

It is possible that many patients avoid complete ophthalmic exams because pupil dilation is slow, reversal sluggish, and vision blurred. Others experience incomplete dilation during exams or prior to surgery when good dilation is essential to successful outcome. Iontophoresis, the application of low-level electrical current to promote traversal of desired molecules across a boundary, has been used for many years and has recently become common in transdermal drug delivery. We now investigate iontophoresis as a method of accelerating drug absorption into the ocular anterior segment. In vivo rabbit studies assessed iontophoresis effects on the performance of dilators and constrictors. 1-mA and 4-mA direct current levels applied for 2-minute durations yielded dilation time-history measurements. Subsequent in vitro tests at a wide range of current densities showed minimal chemical modifications in ocular pharmaceuticals. Drug samples processed through high-performance liquid chromatography (HPLC) pinpointed minimal structural changes. Detailed in vivo rabbit testing is under way. Using 2 dilators and constrictors in crossed testing with 0.5-mA to 1.25-mA current levels and 20-sec to 60-sec durations, we recorded dilation progress by digital photography. Initial studies showed faster, larger dilations and quicker reversal using iontophoresis. Drug testing showed chemical structures remaining constant for clinically useful current levels, < or = 1 mA (< or = 1.25 mA/cm2 current density). Drug pH and HPLC retention times were constant within this range, and resistivity varied linearly as expected for increasing current. Rabbit testing will quantify improved drug speed and efficacy, validate the charge delivery electrode design, and indicate iontophoretic current and duration for further use. Tested ocular drugs showed no degradation when exposed to clinically useful iontophoretic currents. Preliminary results indicate significant time reductions for dilation and reversal, plus increases in maximum dilation. This procedure may aid clinicians by allowing more rapid complete examinations and surgical preparations for patients. Making dilation more convenient will also improve patient acceptance of exams, aiding earlier detection and treatment of ocular disease.     

A rapid method for the separation and analysis of carrageenan oligosaccharides released by iota- and kappa -carrageenase

Based on the improved performances in speed of chromatographic separation on Superdex-type materials (Pharmacia) compared to conventional media such as Sephadex and Bio Gel-type, a rapid size-exclusion chromatography (SEC) method was developed for the separation and analysis of carrageenan oligosaccharides. It was used to evaluate the elution profiles of hydrolysates produced by carrageenases specific for kappa- and iota-carrageenans. Oligosaccharide peaks ranging from di- to dodeca-saccharides were obtained in about 20 min on an analytical scale, whereas preparative runs were completed in a few hours. The method may also be used to monitor polysaccharide degradation.     

A high-performance liquid chromatography method for the assay of cytidine monophosphate-sialic acid synthetase

An HPLC method has been developed for the assay of cytidine monophosphate-sialic acid synthetase (EC 2.7.7.43) using ion-pair chromatography and gradient elution. This procedure permits the assay of alternative substrates and inhibitors of the enzyme and is not subject to the limitation of the colorimetric method. The newly synthesized N-acetyl-9-deoxy-9-fluoro-D-neuraminic acid was found to be a good substrate of the enzyme with a Km of 6.35 mM as compared to 1.84 mM for N-acetylneuraminic acid.     

A method for determination of iododeoxyuridine substitution of thymidine using reversed-phase high-performance liquid chromatography

An assay for thymidine substitution by iododeoxyuridine (IdUrd) using reversed-phase high-performance liquid chromatography (HPLC) has been developed. Three principal steps in this procedure are: extraction of DNA from cell or tissues, hydrolysis of DNA into deoxynucleosides and separation using HPLC. Approximately 1 microgram of DNA was recovered from 10(5) cells by phenol extraction, and subjected to hydrolysis into deoxynucleosides which required a three-stage DNA digestion using enzymes DNAse I. phosphodiesterase I and alkaline phosphatase. The deoxynucleosides were separated on the Microsorb C18 column with isocratic elution; 90-100% of the DNA was recovered as deoxynucleosides on the column. The method was used to determine quantitatively the percent IdUrd substitution of thymidine in Chinese hamster lung cells in vitro and BA1112 rhabdomyosarcoma in WAG/Rij rats perfused with IdUrd. It was possible to determine the thymidine substitution by IdUrd as small as 1% using a few micrograms of DNA. The close correspondence between the percent substitutions determined by HPLC and those determined by radioactive assay using [125I]-labelled IdUrd, confirmed the accuracy of our HPLC method. The HPLC analysis is especially suitable for the determination of percent IdUrd substitution of thymidine in tissue biopsies from animals used in in vivo experiments or humans undergoing radiation treatment.     

Membrane chromatographic method for the rapid purification of vitellogenin from fish plasma

A membrane chromatographic method was developed for the rapid purification of vitellogenin (Vtg) from the plasma of 17beta-estradiol induced loach (Misgurnus angaillicaud atus) and carp (Cyprinus carpio). The time required for the proposed procedure is less then 10 min at a flow-rate of 5 ml/min of the mobile phase, and 0.5 ml of fish plasma could be separated in one cycle. Multistep gradient elution was more suitable for the separation than linear gradient elution. Under optimized conditions, a single Vtg peak can be obtained and its identity was confirmed by SDS-PAGE and gel-permeation chromatography assessment. This method is rapid and easy to operate compared to conventional HPLC and FPLC columns for Vtg separation.     

A miniaturized column chromatography method for measuring receptor-mediated inositol phosphate accumulation

Inositol phosphates (IPs), such as 1,4,5-inositol-trisphosphate (IP(3)), comprise a ubiquitous intracellular signaling cascade initiated in response to G protein-coupled receptor-mediated activation of phospholipase C. Classical methods for measuring intracellular accumulation of these molecules include time-consuming high-performance liquid chromatography (HPLC) separation or large-volume, gravity-fed anion-exchange column chromatography. More recent approaches, such as radio-receptor and AlphaScreen assays, offer higher throughput. However, these techniques rely on measurement of IP(3) itself, rather than its accumulation with other downstream IPs, and often suffer from poor signal-to-noise ratios due to the transient nature of IP(3). The authors have developed a miniaturized, anion-exchange chromatography method for measuring inositol phosphate accumulation in cells that takes advantage of signal amplification achieved through measuring IP(3) and downstream IPs. This assay uses centrifugation of 96-well-formatted anion-exchange mini-columns for the isolation of radiolabeled inositol phosphates from cell extracts, followed by low-background dry-scintillation counting. This improved assay method measures receptor-mediated IP accumulation with signal-to-noise and pharmacological values comparable to the classical large-volume, column-based methods. Assay validation data for recombinant muscarinic receptor 1, galanin receptor 2, and rat astrocyte metabotropic glutamate receptor 5 are presented. This miniaturized protocol reduces reagent usage and assay time as compared to large-column methods and is compatible with standard 96-well scintillation counters.     

A preparative high performance liquid chromatography method for separation of lecithin: comparison to thin-layer chromatography

We have developed a high performance liquid chromatography (HPLC) method to separate lecithin from other phospholipid classes and to obtain lecithin from biologic materials. The separation was performed on a preparative 10-micron Spherisorb column with an optimized solvent system consisting of the following components: acetonitrile, isopropanol, methanol, water, and trifluoroacetic acid. The advantages of this method are the use of an isocratic solvent system limited to about 30 min and the very good separation of the phosphatidyl-choline fraction from the sphingomyelin fraction. Furthermore, the HPLC method has a better recovery rate than the thin-layer chromatography method, and it can be run under automatic control.     

Analysis of mycotoxins in cereals and animal feed using a multi-toxin gel permeation chromatography clean-up method

Gel permeation chromatography has been used to clean up extracts from cereals and animal feeds containing a range of mycotoxins. A mixture of dichloromethane: IM hydrochloric acid, 10:1 by volume is used as the extraction solvent and clean-up is carried out on a Bio — Beads S-X3 column using dichloromethane: ethyl acetate containing a small amount of formic acid as the elution solvent. Chromatographic separation and detection was by HPLC with fluorescence or ultraviolet detection although the choice of detection method is left to the user. The method has been tested for 14 mycotoxins and results are presented for cereals fortified with mycotoxins and for samples naturally contaminated with aflatoxins, citrinin, zearalenone, and ochratoxin A.