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This paper compares high performance capillary electrophoresis (HPCE) and conventional slab electrophoresis in mapping of four closely related plasmids with three different restriction enzymes. The plasmids express full length and truncated forms of a growth factor receptor oncogene product and were digested with HpaII, HaeIII and RsaI. The resulting oligonucleotide fragments were under 2000 base pairs in length, a size well suited to separation by HPCE with linear polyacrylamide as a sieving matrix. Plasmid mapping is an essential tool in biotechnology both for the design of an expression system and for monitoring the stability of the expression system during fermentation. HPCE can yield much higher resolution of oligonucleotides than attainable in conventional agarose gel electrophoretic procedures for plasmid mapping. In the examples described here, the HpaII digests provided the surest identification of individual plasmids in the HPCE analysis and could discriminate among all four plasmids. In conventional slab electrophoresis, however, the RsaI digests provided the best discrimination, although two of the plasmids in this system yielded essentially identical electrophoretic patterns. Hence the optimal restriction enzyme for plasmid mapping applications with HPCE may differ from that selected on the basis of conventional slab gel analysis, and the former technique can provide higher discrimination among related plasmids. The advantages of the HPCE format with respect to speed, low sample consumption and resolution are described.

This paper examines the retention behavior of recombinant DNA-derived human growth hormone (rhGH) in reversed-phase chromatography and its separation from the closely related N-methionyl variant (Met-hGH). It is first shown that retention for rhGH decreases with increasing column temperature when 1-propanol (1-PrOH) is used as organic modifier. On the other hand, retention increases with temperature when acetonitrile (CH3CN) is employed. The differences in behavior for the two organic modifiers could be related to conformational changes in the protein as determined by solution and adsorption intrinsic fluorescence spectroscopy. Specifically, desorption and elution of rhGH using 1-PrOH could be correlated with a solvent-induced conformational change, with retention decreasing with increasing temperature due to the increasing ease of structural alteration. On the other hand for CH3CN the increase in retention correlated with temperature rise was related to a partial structural change yielding a more hydrophobic species. In this case, a surface-driven process is suggested. The work then turned to the separation of rhGH and Met-hGH where it was found for both organic modifiers optimum separation occurred at 45 degrees C and pH 6.5. Separate studies revealed that during the conformational change Met-hGH appeared more hydrophobic than rhGH since protein-protein aggregation was observed at a lower 1-PrOH concentration. It is suggested that this hydrophobic difference, which was optimized under the conditions cited above, resulted in the separation. The study demonstrates the importance of conformational changes in retention behavior and separation of protein samples.

In this study 130 ossicular middle ear transplants were studied. 33 remained in the middle ear for 10 years or longer, one for 21 years. The majority of the grafts show 5% to 40% bone new formation. Absence of vital bone or subtotal host bone replacement is exceptional. The amount of bone new formation appears independent the implantation time. 75% of the grafts show no bone resorption. In 15% sound transmitting might be impaired due to resorption. Grafts with a high degree of bone new formation show less resorption. 21% of the ossicles have signs of inflammation. Inflammation boosts bone turnover with resorption on one hand and new for resorption. Cholesteatoma is the main cause of resorption and inflammation. The critical phase for resorption of the graft are the first two to three years after transplantation. No considerable difference was found between autografts and allografts (homografts). Histological and metabolical properties of the ossicles seem to be responsible for the stability of the graft. Ossicular grafts have a good longterm stability which makes them very suitable for reconstruction of the ossicular chain.

Adsorption-desorption isotherms of bovine beta-lactoglobulin A (beta-lact A) on a weakly hydrophobic stationary phase (C1-ether) were measured by frontal analysis. The adsorption isotherms obtained at different pH were found to be dramatically different in shape, column capacity and desorption reversibility. At pH 4.5, an S-shaped adsorption isotherm was observed whereas at pH 6.0 a Langmuir isotherm was found. In addition, the desorption isotherm at pH 6.0 was found to overlap with the adsorption isotherm, and the adsorption-desorption process of beta-lact A under this condition could be characterized by a fully reversible Langmuir model. The desorption isotherm at pH 4.5, however, did not retrace the adsorption isotherm, resulting in hysteresis loops. A higher aggregate (tetramer) of beta-lact A is shown to be in an equilibrium with the beta-lact A protomer (dimer) at pH 4.5 whereas the dimer alone is predominant at pH 6.0. It is further shown that changes in the absorption coefficient between the adsorption and the desorption cycles for the tetramer at pH 4.5 can account for the hysteresis. The results demonstrate that pH can be a sensitive parameter in protein adsorption isotherm behavior and ultimately the behavior of species in preparative-scale chromatography.

The influence of column temperature on the electrophoretic behavior of myoglobin and alpha-lactalbumin in high-performance capillary electrophoresis (HPCE) is presented. The major effect of temperature is to shorten the analysis time by decreasing the viscosity, but specific temperature effects on the protein migration behavior were also observed. Myoglobin, under high field (350 V/cm), was essentially temperature stable from 20 to 45 degrees C, but at constant current, a second form of myoglobin could be detected at both 214 and 410 nm. The initial form appeared to correspond to the Fe3+ and the second to the Fe2+ oxidation state of the heme iron. The rate of conversion from Fe3+ to the reduced Fe2+ in myoglobin, under given electrophoretic conditions, followed first-order kinetics with a rate constant at 30 degrees C of 304 s-1. A second protein, alpha-lactalbumin type III, demonstrated a conformational transition that resulted in asymmetric peaks and sigmodial mobility plots versus temperature in the transition region.

The effect of metal ion [Ca2+ and Zn(NH3)4(2+)] on the unfolding kinetics of bovine-alpha-lactalbumin (alpha-LACT) on a weakly hydrophobic chromatographic surface (ethyl polyether phase bonded on porous silica, C2 ether) has been studied using surface intrinsic fluorescence spectroscopy and liquid chromatography. Chromatographic results on the C2 ether phase revealed two peaks for alpha-LACT, the first being the folded and the second an unfolded conformation, as determined by fluorescence spectroscopy. The retention time for the second peak was found to depend on the specific metal additive in the mobile phase. Fluorescence studies showed a slow change in emission maximum from ca. 330 nm to 350 nm and a 5-fold increase in emission intensity for the adsorbed protein in the unfolded state. By following the fluorescence emission intensity at a given wavelength during the unfolding process, biphasic kinetics were observed with the kinetic constants depending on the specific metal-ion additive. In addition, solution refolding rates of the desorbed, unfolded species were measured and found to be consistent with literature refolding rate constants.

This paper demonstrates the potential of capillary gel electrophoresis with laser induced fluorescence detection as a tool for DNA sequence determination. Both synthetic oligonucleotides and single-stranded phage DNA were utilized as templates in the standard chain termination procedure. Primer molecules were tagged at the 5' end with the fluorescent dye, JOE. First, baseline resolution of a dA extended primer from 18 to 81 bases long, a total of 64 fragments, was observed. A second synthetic template was designed to yield alternating stretches of dA and dT extensions of the primer. Thirdly, the sequence reaction products from a synthetic oligonucleotide template containing all four bases was analyzed in four independent runs, one for each of the four base-specific reactions. In all cases, the expected number and patterns of peaks were observed by capillary gel electrophoretic analysis. Finally, separation of sequence reaction products generated with single-strand M13mp18 phage DNA as template exhibited baseline resolution of fragments differing in length by a single nucleotide and from 18 to greater than 330 bases total length.

This paper presents results on the separation of DNA restriction fragments by high performance capillary electrophoresis (HPCE). Capillaries containing polyacrylamide with low amounts of crosslinking agent (i.e. 0.5% C) were first studied. The greater molecular accessibility offered with columns of low crosslinking, relative to higher crosslinked gels (e.g. 5% C), permitted high efficiency separations of double stranded DNA fragments up to 12,000 base pairs in length. Capillaries containing no crosslinking agent, i.e. linear polyacrylamide, were then examined. Ferguson plots (i.e. log mobility vs. %T) were used to assess the size selectivity of linear polyacrylamide capillaries. In another study, it was determined that the relative migration of DNA species was a strong function of applied electric field and molecular size. Lower fields yielded better resolution than higher fields for DNA molecules larger than about 1000 base pairs, albeit at the expense of longer separation time. Based on these results, we have examined pulsed field HPCE and have demonstrated the use of this approach to enhance separation.

The kinetic mechanism of unfolding of calcium depleted bovine alpha-lactalbumin adsorbed on two weakly hydrophobic chromatographic surfaces, methyl- and ethyl-polyether phases bonded to porous silica, with a solution phase of 3 M ammonium sulfate at pH 6.3, has been determined using intrinsic fluorescence and liquid chromatography (LC). The adsorbent has been packed into quartz flow cells which are used for both fluorescence measurements and as a microcolumn for LC. The LC measurements revealed two peaks for alpha-lactalbumin on both phases, the first being folded and the second unfolded. The rate of unfolding was measured to be 1.75.10(-3) min-1 on the Cl-ether and 7.42.10(-3) min-1 on the C2-ether phase. Fluorescence studies revealed a slow change in emission maximum from ca. 330 nm to 350 nm and a 4-fold increase in intensity for the protein adsorbed on the two supports. Variation of fluorescence intensity at a given wavelength revealed biphasic kinetics in which the rate law on the surface was deduced as F in equilibrium X----U, where F is the folded form, U an unfolded form and X an intermediate. The normalized emission spectra of the three species were calculated and it was found that there was approximately a 20-nm-red shift in the position of the maximum from F to U. The emission maximum for X was close to U on both columns; however, the normalized intensity for X was between F and U. Activation enthalpies and entropies were determined from the temperature dependence of the microscopic rate constants. The formation of the intermediate on the C1-ether phase was entropy driven whereas on the C2-ether phase it was enthalpy driven. Finally, the solution refolding rates of U desorbed from the two supports were found to be identical. The differences observed in the surface kinetics of unfolding on the two supports are related to the hydrophobic differences of the adsorbents.

A series of standard small globular proteins in different unfolded states was studied by gradient reversed-phase liquid chromatography. The retention parameters Z [slope of log capacity factor (k') vs. log molar concentration of organic modifier, 1-propanol, in the mobile phase] and log I (the value of log k' at 1 M 1-propanol) were derived from gradient retention data. Each protein in four different conformational states, i.e., folded, chromatographic surface-unfolded, urea-unfolded and disulfide-bridge reduced-unfolded, showed a variation of 10-fold in Z and up to 10(12)-fold in I values. For the different states of all the proteins studied, the order of Z and I values was as follows: folded much less than surface-unfolded less than urea-unfolded less than reduced-unfolded. The differences in the values of the coefficients suggest, in agreement with literature reports, that proteins with their disulfide bridges cleaved have the largest degree of unfolding. In addition, the Z and I values and solution refolding kinetics all suggest that chromatographic surface-unfolded proteins have a lower degree of unfolding than their urea-unfolded forms. It was also found that an additional chemical cross-link in lysozyme caused a significant decrease in the first-order rate constant of the surface-induced unfolding process.

The mass spectrometric properties of a series of model ion pairs were examined. In the cases studied it was possible to vaporize the ion pair consituents and to produce spectra corresponding to those of the unpaired materials. These findings offer a convenient means for derivatizing certain ionic compounds and demonstrate the feasibility of analyzing ionic species by on-line liquid chromatography-mass spectrometry.

This paper examines the use of wide-pore silica-based hydrophilic ether-bonded phases for the chromatographic separation of proteins under mild elution conditions. In particular, ether phases of the following structure identical to Si-(CH2)3-O-(CH2-CH2-O)n-R, where n = 1, 2, 3 and R = methyl, ethyl or n-butyl, have been prepared. These phases can be employed either in high-performance hydrophobic-interaction or size-exclusion chromatography, depending on mobile phase conditions. In the hydrophobic-interaction mode, a gradient of decreasing salt concentration, e.g., from 3 M ammonium sulfate (pH 6.0, 25 degrees C), yields sharp peaks with high mass recovery of active proteins. In this mode, retention can be controlled by salt type and concentration, as well as by column temperature. In the size-exclusion mode, use of medium ionic strength, e.g., 0.5 M ammonium acetate (pH 6.0) yields linear calibration of log (MW[eta]) vs. retention volume. Even at 0.05 M salt concentration, no stationary phase charge effects on protein elution are observed. These bonded-phase columns exhibit good column-to-column reproducibility and constant retention for at least five months of continual use. Examples of the high-performance separation of proteins in both modes are illustrated.

High-performance capillary electrophoresis (HPCE), the instrumental approach to electrophoresis, is a method undergoing rapid development at the present time. There is a high expectation that HPCE will become a widely applicable tool within the biochemical community. This review presents principles and instrumentation followed by typical applications of HPCE in the biological area. In the instrumentation area, particular attention is paid to current and future detectors, including laser-induced fluorescence and HPCE-mass spectrometry. In the applications section, both peptide or protein and oligonucleotide high-resolution separations are described for open-tube and polyacrylamide gel capillary columns. The use of isoelectric focusing and electrokinetic chromatography to manipulate separation is also presented. Future directions of methodology are suggested, and it is predicted that in the next few years HPCE will become a complementary tool to liquid chromatography and slab gel electrophoresis.

This paper explores the influence of mobile-phase and temperature effects on the gradient elution reversed phase chromatographic behavior of proteins. Using LiChrospher SI 500, bonded with n-butyl groups and a gradient in 1-propanol, 10 mM H3PO4, rapid separation and high mass recovery were obtained for a series of globular proteins. This protein separation and recovery are compared to those obtained when acetonitrile and acetonitrile plus 10 mM H3PO4 are used as eluting gradient solvents. In general, acetonitrile yielded lower recovery than 1-propanol, particularly for the more hydrophobic proteins, e.g., ovalbumin. For all three gradient solvents, little difference was observed in retention or recovery when the n-alkyl chain of the bonded phase varied. On the other hand, relative to the n-alkyl phases, a significantly lower retention of all proteins was found on more hydrophilic phases, e.g., cyano and nonendcapped n-butyl, when acetonitrile was the organic modifier, while in the case of 1-propanol, no retention difference was observed. Thus, column comparisons depend on the protein/mobile-phase combinations examined. The role of column temperature was also studied, and it was found that for certain proteins dramatic changes in peak shape occurred as a function of temperature. The influences of ionic strength and salt type were also studied. Protein mass recovery was shown to decrease with an increase in salt concentration; moreover, perchlorate was shown to have a larger effect in this regard than phosphate. In addition, salt concentration and type were found to influence peak shape greatly in certain cases. The results indicate the strong influences of mobile phase and temperature on chromatographic behavior, and some of the options available when this behavior is not satisfactory. Several protein separations are presented illustrating the power of the reversed phase approach.

As a continuation of previous studies, we present in this paper measurements on the kinetics of denaturation of papain, soybean trypsin inhibitor and lysozyme on n-butyl-bonded silica gel surfaces used in reversed-phase liquid chromatography (RPLC). In all cases, native and denatured peaks widely separated from one another are observed. The rate constants for denaturation or unfolding are determined by the measurement of the peak area of the native protein as a function of the incubation time that the species spends on the bonded-phase surface. The results reveal that a slow denaturation step occurs with a half-life of ca. 15 min. In addition, studies of denaturation as a function of the amount of 1-propanol in the initial mobile phase suggest an additional unfolding step when the protein comes in contact with the bonded-phase surface. The extent of this latter step decreases as the concentration of 1-propanol increases, further suggesting that 1-propanol sorption on the bonded stationary phase may play a role in this behavior. Other studies are conducted with alpha-chymotrypsinogen, in which injection is made after the start of the gradient. The extent of denaturation is observed to be a function of the organic modifier employed. The results of this paper provide insight into the denaturation process in RPLC and suggest approaches to minimize this behavior.

The applicability of high-performance hydrophobic interaction chromatography using newly developed silica-based ether-bonded phases is demonstrated in the purification of the rat liver enzymes carbamoylphosphate synthetase I and ornithine transcarbamoylase from crude mitochondrial extracts. As a result of the mild adsorption/elution conditions in this high-performance chromatographic mode, the enzymes are recovered in 20 min with 3- to 15-fold increases in specific activity. Since the enzymes are labile and may aggregate in solution, in one case up to Mr 330,000, this rapid purification demonstrates the potential of hydrophobic interaction chromatography in complex biological systems.

Open-tube capillary electrophoresis has been applied to the separation of restriction fragments of DNA with a Tris-borate buffer containing 7 M urea and 0.1% sodium dodecyl sulfate. The importance of sample pretreatment and of the injection of heated samples has been demonstrated. In one separation, a DNA restriction fragment mixture from 72 to 23,130 base pairs (DRIgestTM III) (molecular weight range from 4.6.10(4) to 1.5.10(7] has been electrophoresed in 10 min on a column of 15 cm effective length. Over 600,000 plates have been obtained for individual peaks. Several of the peaks have been identified, by spiking slab gel electrophoretically purified components. Other examples of restriction fragment separations are illustrated in this paper. The results of this study when further validated with full characterization of individual species, open up the possibility of rapid restriction enzyme mapping.

Picomole amounts of oligodeoxynucleotides [polydeoxyadenylic acids, (dA)40-60] were baseline resolved and analyzed in less than 8 min by high-performance capillary electrophoresis with polyacrylamide gels. In addition, fast analysis of a crude 70-mer oligodeoxynucleotide and a slab gel-purified 99-mer oligodeoxynucleotide was accomplished, demonstrating the ability of high-performance capillary electrophoresis to characterize rapidly synthesized oligonucleotides. Besides analytical separations, 800 ng of a primer (20-mer) was isolated in less than 20 min. The purified species was collected in water and subsequently used as a probe in a standard dot-blot analysis. The use of high-performance capillary electrophoresis for the analysis and purification of a variety of biopolymers is simple, rapid, and has the potential for automation.

This paper explores the role of association on the adsorption isotherms of beta-lactoglobulin A on a weakly hydrophobic stationary phase at 4 degrees C and mobile phases of 0.85 M and 1 M ammonium sulfate, pH 4.5. The isotherms, obtained by frontal analysis, show an S-shape and the corresponding Scatchard plots indicate positive cooperativity. The slopes and intercepts of the Scatchard plots at low solute concentration are analyzed in terms of two species--a promoter and a higher order stronger adsorbing species. An explicit equation of the isotherm is developed based on this model, and this expression is shown to reproduce the isotherm shape using the appropriate derived parameters. It is further shown from this equation that a Langmuir-shaped adsorption isotherm can be obtained if the higher order associate or aggregate binds weaker to the support than the promoter. These results indicate that protein-protein interactions and the formation of associates can play a significant role on the shape of the isotherm and ultimately on the behavior of the species in preparative scale chromatography.

High-performance ion-pair partition chromatography is shown to be a versatile, efficient method for separating sulfonamides. For a group of fourteen sulfa drugs varying widely in pKA and hydrophobicity, the effect of mobile phase composition, counterion composition, pH, and ionic strength on their ion-pair partition chromatographic separation using tetrabutylammonium as the counterion and n-butanol-n-heptane as the mobile phase is shown. Wide variation in k' and a is possible by changing these parameters. Silica columns coated with buffered aqueous solutions of tetrabutylammonium sulfate resulted in efficiencies of 4000-6000 theoretical plates per 25 cm. These columns are stable for long periods of time, and can be stripped and re-used in the adsorption mode with little or no loss in efficiency. Several chromatograms are presented in order to illustrate the performance of ion-pair partition chromatography.

High-performance separations of proteins, based on immobilized-metal affinity chromatography (HPIMAC), are described. The stationary phase consisted of iminodiacetic acid (IDA) chelate groups, bonded to small particle, wide pore silica gel by means of a polyether hydrophilic leash. After loading the column with metal, retention of proteins was achieved by protein-metal complexation at high concentrations of sodium sulfate. Elution was accomplished by addition of competitive complexing ligands, such as ammonia at constant pH, and/or by a decreasing pH gradient of a specially designed buffer system to maintain buffer capacity constant throughout the gradient. Selective separations, based on differences in the number of histidine residues present on the surface of the proteins, are described. The application of HPIMAC in the separation and purification of structurally similar proteins is presented. The potential application of IDA columns in three chromatographic modes (HPIMA, hydrophobic interaction, and cation exchange) is also described.

Whenever proteins are found in environments different from those provided by physiological conditions, structural alterations can occur which can dramatically affect their adsorption and chromatographic behavior. The resultant behavior is often kinetically controlled and thus dependent on such factors as contact time of the protein with the adsorbent surface. Examples are given of the appearance of multiple peaks from seemingly pure species, as a result of these structural changes. In one case (papain in reversed-phase LC), multiple peaks are shown to arise from different conformational states. In a second case (beta-lactoglobulin A in hydrophobic interaction chromatography), a series of three peaks is a result of self-association or aggregation. Finally, recent work on an examination of structural changes of proteins on chromatographic supports, by means of intrinsic fluorescence and HPLC, is presented. The value of these studies for the elucidation of the retention mechanism in HPLC and the assessment of purity of proteins is demonstrated.

N-Acetyl-N,O,S-permethylated derivatives of oligopeptides were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) using a moving belt interface. A heated-gas nebulizer was employed for sample deposition, thus permitting the effective use of a water-methanol gradient covering the range from 5% to 95% water at mobile phase flow-rates of 0.5 ml/min. We demonstrate in this paper that it is possible to sequence the octapeptide derived from the C-chain of glucagon by HPLC-MS analysis of a permethylated enzymatic hydrolysate of this peptide using overlap information from the mass spectral patterns. Moreover, it is shown that peptides not readily amenable to analysis by gas chromatography-MS can be analyzed using this approach. Preliminary results suggest that N-acetyl-methyl ester derivatives of oligopeptides may in specific cases also be a useful alternative for HPLC-MS analysis of complex oligopeptide mixtures.

This paper is a continuation of the evaluation of hydrophilic ether columns for the separation of proteins by hydrophobic-interaction chromatography. In this approach, linear salt gradients of decreasing concentration of ammonium sulfate yield sharp chromatographic peaks with high mass recovery and maintenance of biological activity. Mild adsorption conditions are indicated by the minimal changes in chromatographic peak area of native proteins as a function of contact time by the biopolymer with the stationary phase. Further evidence of minimal kinetic processes under the chromatographic conditions are seen in the constancy of isocratic retention with mobile phase flow-rate and sample load up to 2 mg. Based on the well-behaved chromatographic characteristics, we have explored gradient optimization in terms of the Snyder model for gradient elution. It is shown that changes in retention, peak capacity and peak height follow predicted gradient time dependencies. Moreover, the influence of particle diameter and column length are found to be in agreement with expected behavior, based on the model. As a consequence of the agreement, prediction of conditions for optimum separation for a particular problem are possible. Other studies examine the influence of specific ion effects, e.g., Mg2+ binding to protein, which can override retention based on predicted surface tension behavior. Some characteristics of the ether column for hydrophobic-interaction chromatography are shown, e.g., column stability at pH 8 and sample capacity.

A high-performance liquid chromatography method is presented for simultaneous analysis for procainamide and N-acetyl procainamide in plasma or serum. The procedure involves internal-standard addition, organic extraction, and separation on a reverse-phase column. The detection limit for procainamide is 0.1 mg/liter and the calibration plot is linear to at least 30 mg/liter. Comparison with a colorimetric assay for procainamide gave a correlation coefficient of 0.989. We checked for interference by a large series of appropriate drugs, and found none, nor did icteric or lipemic sera present problems.

An automated high-performance liquid chromatographic column-switching system is presented for the analysis of antiepileptic drugs in serum. Initial results show that a reversed-phase extraction column works best overall when fitted with screens versus frits, and when packed with porous 30-micron particles as opposed to a pellicular packing of similar size or with smaller porous particles. The continuous analysis of primidone for over 2000 serum samples is achieved at a rate of twelve samples per hour with a single analytical column. An analogous boxcar high-performance liquid chromatographic system is also assembled and used to analyze two of four injected antiepileptic drugs at a rate of 40 samples per hour. For 1000 of these analyses, the coefficient of variation is 1% without an internal standard.

This paper is a continuation of an examination of the conformational effects of ribonuclease A (RNase A) in reversed-phase liquid chromatography. RNase A is a particularly good example of reversible conformational refolding during chromatographic elution. Absorbance ratio measurements with a photodiode-array detector are consistent with a two-state model in which an early eluted broad band is associated with the folded or native state and a late eluted sharp band is associated with an unfolded state. By varying the mobile phase flow-rate while maintaining the mean capacity factor (k') of the protein in gradient elution constant, the apparent rate constant of refolding in the mobile phase was measured and found to be comparable with that reported in the literature. It has been further shown that, as the gradient steepness parameter b or mean k' value is altered, the apparent rate constant also changes, as a result of variation in mobile phase composition. The apparent rate constant as a function of temperature was also found to reach a maximum at 20 degrees C. Based on the results of this model system, it is possible to predict optimum conditions for elution of species with sharp elution peaks when reversible refolding takes place in the column.

We have studied the conformational behavior of alpha-lactalbumin (alpha-LACT) in hydrophobic interaction chromatography (HIC). Retention characterization in terms of Z (slope of plot of ln k' vs. ln phi B, where k' is the capacity factor and phi B is the volume fraction of mobile phase B) has been explored, and the relationship of Z to other slopes, such as S (slope of the plot of ln k' vs. phi B) has been derived. The reasons for the sensitivity of Z to conformational change are discussed. The enhanced broadening of alpha-LACT in a temperature transition region of conformational change has been studied by spectral analysis using on-line photodiode array detection. The influence of Ca2+ and Mg2+ addition to the mobile phase is further explored. Since alpha-LACT is a calcium binding protein, addition of this metal leads to stabilization, i.e. higher column temperatures are required for conformational change. On the other hand, addition of Mg2+ appears to destabilize the protein. We have explored the use of a more hydrophobic support, C2-(ethyl) ether phase, for the elution of alpha-LACT. In this case, two widely separated peaks are observed. By spectral analysis the first peak is shown to be native and the later eluted, broad second peak to be an unfolded mixture of species. As previously observed in reversed-phase liquid chromatography, the second peak grows at the expense of the first, as the column temperature is raised. The second peak also grows as the contact time of the protein with the surface increases. This behavior can be ascribed to the conformational change of alpha-LACT in the column, the late eluted species under the second peak binding significantly more strongly to the phase than the native peak. Reinjection of the late eluted fraction reveals that reformation of the native species takes place in solution within 30 min. As before, addition of Ca2+ reduces the extent of unfolding under any specific condition. These results add further to our understanding and ability to control conformational changes in high-performance liquid chromatography.

High-performance capillary sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has been developed for the separation and molecular weight (MW) determination of peptides and proteins. In this work, acrylamide was polymerized in fused-silica capillaries of 75 microns I.D. and 10 or 20 cm length. On-line UV detection was employed by placing the capillary in the optical path of the detector. Rapid separations of myoglobin fragments and of a mixture of standard proteins were demonstrated with efficiencies of the order of 40,000 plates for a column, 20 cm in length. Linear plots of log MW vs. mobility for the SDS protein complexes were observed. Variation in the percentage monomer composition (% T) of the polyacrylamide yielded the expected common intercept of log mobility vs. % T, confirming the size separation mechanism in the gel capillaries. The separation of the A and B chains of insulin with a 10-cm column in less than 10 min was also demonstrated.

A methodology involving equilibrium dialysis and gas chromatography has been developed to measure concurrently the concentration of the dialyzable (i.e., free) fractions of thyroxine and 3,5,3'-triiodothyronine in serum. The electron capture response of the N,0-diheptafluorobutyryl methyl ester derivatives of these hormones allows their detection and quantitation in the picogram range (detection limit approx. 0.2pg). Purification of the dialysate is achieved using a cation-exchange column which is pretreated with diiodothyronine. Adsorption problems on the glassware are eliminated by silanization. The methodology also resolves 3,3',5'-triiodothyronine (reverse T3).

This paper examines the reversed-phase liquid chromatographic behavior of ribonuclease A (RNase) using an n-butyl chemically bonded phase and a gradient of 10 mM H3PO4 and l-propanol. At a column temperature of 25 degrees C, a broad band followed by an overlapped late-eluting sharp peak is observed. As the temperature is raised, the sharp peak grows at the expense of the broad band until at 37 degrees C, only a single narrow-eluting band is found. Using an absorbance ratio of A288/A254, it is demonstrated that the broad band represents a folded or native state of RNase and the late-eluting band a denatured state. Based on postcolumn absorbance ratio changes in the denatured state as a function of time and the known behavior of the protein, reversible refolding or renaturation is proposed to take place in solution. RNase is denatured upon adsorbing to the bonded phase, and upon migration down the column, reversible refolding takes place in the mobile phase. The relaxation time for native state formation is assumed to be comparable to the time spent by RNase in the mobile phase. As temperature is raised, both the native and denatured states exist at equilibrium in solution, thus slowing the refolding process, until at 37 degrees C only the denatured peak appears. Changes in peak shape with flow rate provide further evidence for this model. The use of HCl or H2SO4 instead of H3PO4 yields similar results except that the temperature at which only the denatured peak is observed follows the order of salt stabilization of the native state.

The thermal behavior of a series of standard proteins in hydrophobic interaction chromatography on a previously developed weakly hydrophobic ether-bonded phase column has been studied. Depending on the temperature and protein, conformational changes can occur in the chromatographic system. Methods for recognizing these conformational effects are presented, including retention and peak width variations with temperature, and Z values (the slope of the plot of log k' vs. log %B solvent. The Z value is shown to be a general index characterizing protein retention as a function of salt concentration. In addition, on-line UV spectroscopic analysis, (absorbance ratios and second derivative spectroscopy) with a photodiode array detector, is shown to corroborate chromatographic trends. Lysozyme maintains a stable conformation over the temperature range 10-40 degrees C, whereas beta-lactoglobulin A has a conformational transition between 25 degrees C and 40 degrees C. Calcium-depleted alpha-lactalbumin, a rather labile species, maintains a stable conformation up to ca. 20 degrees C, and then undergoes structural changes. Finally, cytochrome c appears to be relatively stable up to ca. 65 degrees C. Since conformational changes for this protein occur at ca. 35 degrees C on more hydrophobic phases, the extent of hydrophobicity of the stationary phase is important for maintenance of the native state. Based on this work, hydrophobic-interaction chromatography at sub-ambient temperatures appears promising.