HPLC of Proteins, Peptides, It is the practice in ANALYTICAL
to dispense with the first two words in "high-performance liquid chromatography" (HPLC), the rationale being that separations in the high-performance mode so dominate the old open-column mode that the words "high performance" are superfluous. This assumption is not correct in biochemistry. Although it shows great promise, HPLC is still a minor technique for the separation of biopolymers. For the sake of clarity, it is necessary to differentiate between column liquid chromatographic separations carried out with gels of low mechanical strength in low-pressure glass and plastic columns and those achieved with rigid packing materials in high-pressure systems at high-mobile-phase velocities. Samples of biological origin may contain 2000 or more constituents. Unfortunately, HPLC technology has not advanced to the point that it is realistic to think of resolving all these components in a single column. Purification of biological macromolecules is still a multiple-step or multiple-column process. Since the thrust of this REPORT is directed toward chromatography, the separation properties of various classes of compounds by highperformance size exclusion chromatography (SEC), ion exchange chromatography (IEC), reversed-phase chromatography (RPC), and liquid affinity chromatography (LAC) will be discussed rather than specific purification schemes or systems. CHEMISTRY
Polypeptides By far the greatest amount of activity in HPLC of biopolymers has been with proteins and peptides. This reflects the broad interest and involvement of the life sciences community
with polypeptides during the decade of the seventies, when research on HPLC of biopolymers began. Peptides. It may be said without exaggeration that high-performance RPC has revolutionized peptide chemistry. RPC was first applied to the purification of synthetic peptides, where it provided a new standard of purity: "pure by HPLC." Gross impurities were revealed in synthetic hormones, antigens, and peptides used in the health sciences. The technique has now become an integral part of the production of synthetic peptides. More recently RPC has been applied to the purification of natural peptides, often in analytical columns (0.4 X 30 cm). Such small columns may be used in preparative separations because the amount of physiologically active peptides in tissue is small. Recovery of biological activity after RPC is quite good with most low-molecular-weight peptides. Although peptides may have secondary and tertiary structure, their small size increases the probability of their renaturation after exposure to organic solvents in the RPC system. In some of the more imaginative work, dilute tissue extracts are pumped directly onto the RPC column where peptides, proteins, and many other materials accumulate. Subsequent gradient elution and biological assays of eluent fractions reveal the location of the desired active peptides. Since columns are easily overloaded by this technique, rechromatography under identical conditions yields further purification. Another strategy is rechromatography with the same mobile phase on a different RPC column since selectivity may vary between supports in RPC columns (1). Ion-pairing agents also have been used effectively to vary selectivity (1-3). When one considers
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0003-2700/83/A351-1298 $01.50/0 © 1983 American Chemical Society
Report Fred Ε. Regnier Department of Biochemistry Purdue University Lafayette, Ind. 47907
and Polynucleotides that phosphoric, acetic, trifluoroacetic, heptafluorobutyric, formic, and hy drochloric acids—in addition to bicar bonate, pyridinium acetate, triethylammonium phosphate, and mobilephase pH—all change the selectivity of an RPC column for peptides, it is easily seen that a single RPC column may be used to purify a peptide by rechromatography. Another use of RPC is in protein structure elucidation. Protein struc ture is determined by fragmenting the polypeptide either by proteolysis or chemical cleavage followed by purifi cation and sequencing of the peptide fragments. In the case of large pro teins such as thyroglobin, the protein digest may have more than 100 pep tide fragments as shown in Figure 1. The largest problem with protein di gests has been recovery of the more hydrophobic peptides; 80% or more of the amino acid residues in some pep tides may be hydrophobic. Recovery of hydrophobic peptides is strongly in fluenced by both the mobile phase and the support matrix onto which the alkyl silane is bonded (1,4,5). Sup port pore diameter has also been im plicated, but its importance in the res olution and recovery of peptides is probably overrated. Preliminary fractionation of protein digests has usually been carried out on G-50 Sephadex columns with a formic acid mobile phase. Although many of the peptides are very hydrophobic as noted above and fractionations are often not on the basis of molecular size, preliminary SEC fractionation serves to reduce the complexity of subsequent RPC chromatograms (4). High-performance SEC columns eluted with acidic mobile phases contain ing some organic solvent are also ef fective in preliminary fractionations
U).
The most popular mobile phase for RPC of polypeptides has been a gradi ent of 0.1% trifluoroacetic acid (TFA) in water to 0.1% TFA in acetonitrile or propanol (4,5). TFA with an organic solvent such as methanol, propanol, or acetonitrile is an excellent solubilizing agent for peptides, allows detection in the 230-240-nm range, and may be evaporated to give peptides free of mobile phase. This last property is particularly important in sequencing and biological assays. Although ion exchange chromatog raphy of peptides has been reported to be of great utility in several cases, it is not widely used in peptide purifica tion. Biologically active proteins. The functional properties of many biologi cally active proteins, for example, en zymes, hormones, antibodies, and transport proteins, are often due to
their unique structures. The fact that SEC and IEC separations may be car ried out at nearly physiological condi tions makes them ideally suited to this class of polypeptides. Both SEC and IEC routinely give 80-100% recovery of biological activity. Enough different pore sizes of high-performance SEC columns are now available that col umns may be found for the fraction ation of polypeptides from 10 to 1000 kilodaltons (KD). An example of the use of high-performance SEC in the separation of calf cortical and nuclear water-soluble lens proteins is shown in Figure 2 (6). HPSEC columns may be viewed as the equivalent of the best conventional gel-type columns in all cases except for the separation of very basic or hydrophobic proteins. The slight anionic and hydrophobic char acter of commercial HPSEC columns will cause slight deviations from ideal
Figure 1. The reversed-phase chromatogram of a tryptic digest of human thyroglobulin Elution was achieved with a linear gradient of 100 mM ammonium bicarbonate ranging to 50% acetoni trile in 100 mM ammonium bicarbonate over 300 min. Adapted from Reference 7 ANALYTICAL CHEMISTRY, VOL. 55, NO. 13, NOVEMBER 1983 · 1299 A
FOR OPTICAL PURITY ANALYSIS AND SEPARATION OF OPTICAL ISOMERS A Proven HPLC Method... Developed by Pirkle*, the method is: • Sensitive... Only nanograms of sample required •Unequivocal*..Trace amounts of one enantiomer determinable in presence of great excess of the other • Insightful... Chiral separations observable even in complex mixtures • Preparative... Up to 100 mg per pass with 10 mm I.D. column
From A Reliable Column Source... Regis is a pioneer and leader in developing HPLC columns based on the Pirkle* concept of chiral separation. Regis chiral columns are: •Efficient...Average 40,000 plates per meter • Versatile... Separate many classes of aromatic compounds such as alcohols, hydroxyls, sulfoxides, hydantoins, and succinimides Figure 2. High-performance size exclusion chromatography of calf cortical and nuclear water-soluble lens proteins Elution profiles from bottom to top: differential refractive index (DRI) detection of nuclear crystallins; DRI detection of cortical crystallins; and low-angle laser light scattering (LALLS) detection of cortical extract (note that the signal in the HM- and alpha-crystallin region is attenuated by a factor of 20). Adapted from Reference 6
SEC behavior unless mobile-phase ad ditives are used to control adsorption. Protein fractionation by HPIEC has increased five- to tenfold in the past two years. Gradient elution HPIEC is attractive because in addi tion to high recovery it has resolution comparable to polyacrylamide gel electrophoresis and 10 times the load ing capacity of SEC. For example, a 0.41 X 25-cm column does not begin to overload until samples >10 mg are used (5). Even in the overloaded con dition produced by a 50-mg sample, the purification of ovalbumin from bo vine serum albumin on this small col umn will still be satisfactory. There is now sufficient literature on HPIEC to make the broad observation that it is at least 10 to 60 times faster than con ventional gel-type columns with com parable or superior resolution and re
covery. An example of a high-perfor mance anion exchange separation of proteins on a weak ion exchange col umn is seen in Figure 3 (7). Although used less extensively, high-performance LAC also has been effective in the fractionation of biolog ically active polypeptides. Retention in LAC is based on the biospecific as sociation of a protein with a substance immobilized on a column that mimics a substrate, effector, receptor, antigen, or some other ligand with which a pro tein naturally associates. Elution is achieved either by the addition of a substance to the mobile phase that competes with the stationary phase for binding sites on the protein or by changing mobile phase conditions suf ficiently to alter protein structure and diminish the strength of the associa tion (8). Immobilized adenine mono-
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 13,
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nucleotide has been shown to retain both oxidoreductase and kinase enzymes. This is because both of these classes of enzymes have adenine binding sites for association with their respective coenzymes, nicotinamide adenine dinucleotide and adenosine triphosphate. Isoenzymes can even be separated with these cofactor-mimicking columns (8, 9). A second technique for the purification of proteins at the adenine binding site is the use of LAC columns with immobilized triazine dyes (10,11), particularly Cibacron Blue F3G-A. Triazine dyes also associate with the adenine binding site of proteins but with less specificity than adenine. Unfortunately, recovery of enzyme activity from the LAC triazine columns is only 25% of that achievable with comparable gel-type supports (10). Lowe et al. indicate that the value of triazine dye columns is that they are more versatile and less expensive; this, however, is achieved at the expense of selectivity and recovery. LAC also may be kinetically different from SEC and IEC. The binding of an antigen by an immobilized antibody may be represented by an apparent binding constant whose value is flow rate dependent (12). Basically the same observation has been made for the binding of proteases to immobilized soybean protein (13). Dissociation of the protein ligand complex also may be very slow. Both resolution and column-loading capacity are strongly influenced by support pore diameter (14). Walters has recommended the use of either 60-Â pore diameter supports that prevent the penetration of macromolecules or 4000-Â pores that allow rapid diffusion within the pore network (14). With properly designed supports and operating conditions, LAC can afford very rapid analyses with very high selectivity and throughput. RPC has been of little use in the fractionation of enzymes. Recovery of enzyme activity from RPC columns is notoriously difficult because of the necessity of using organic solvents for elution. Although new techniques will emerge for renaturing proteins, it is doubtful that RPC will ever be a general technique for fractionating enzymes unless some way is found to reduce the hydrophobicity of the current generation of columns. Membrane proteins. Approximately 20-80% of cell membranes may be protein. These proteins can be divided into two broad classes: peripheral membrane proteins associated with the outer surface of the membrane and integral membrane proteins that are either totally engulfed in the membrane, span it partially, or span the membrane completely. The lipophilic
Figure 3. High-performance anion exchange chromatography of carbonic anhydrase, ovalbumin, and soybean trypsin inhibitor The column was eluted at 0.5 mL/min with a 40-min linear gradient ranging from 0.02 M tris (pH 8.0) to 0.5 M NaCI in 0.02 M tris (pH 8.0). Adapted from Reference 7
environment within the lipid bilayer of the membrane dictates that proteins situated in the membrane must be much more hydrophobic than the average water-soluble protein found in the cytosol. In fact, it is known that some integral membrane proteins and viral coat proteins are composed of 75% or more of hydrophobic amino acids (15). Finding a solvent for these proteins has been difficult. As Khorana et al. noted in a recent paper "the straightforward application of current methods for sequencing water-soluble proteins to large hydrophobic membrane proteins such as bacteriorhodopsin is not feasible" (16). On initial examination of the problem, it would seem that RPC is the only chromatographic technique suited to the separation of such hydrophobic materials. Actually, both RPC and IEC have been used in the purification of membrane proteins (17). Since formic acid is a superior solubilizing agent for peptides and has long been used as a mobile phase in their separation on Sephadex columns, Khorana
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et al. were led to use formic acid and ethanol as the eluent for cyanogen bromide cleavage fragments of bacteriorhodopsin (16). Fragments of this protein with molecular weights up to 19 KD and 70% hydrophobic residues were successfully chromatographed on RPC columns using ethanol gradients of 5% formic acid. More recently, formic acid has been used in conjunction with propanol for RPC of proteins (15). Proteins can be induced by 60% formic acid to elute from columns with 50-80% less organic solvent than required when 0.1% TFA is used in the mobile phase. The principal problem with formic acid is that it is sufficiently acidic to initiate hydrolysis of some sensitive proteins and to esterify hydroxyl groups. A second approach to the separation of membrane proteins has been the use of either nonionic detergents or organic solvents with IEC. Coat proteins of sendi virus and bovine viral diarrhea virus have been separated on anion exchange columns using 0.1% Triton X-100 and 0.2% Berol additives, respectively (17). An alternative procedure has been to solubilize hydrophobic proteins with organic solvents during the course of IEC. Chloroplast proteins have been fractionated on an anion exchange column eluted with chloroform:methanokwater (3:3:1) to which a 25-min gradient of 0-20 mM ammonium sulfate was applied (17). Elution according to increasing ionic character was observed. Other proteins. A number of other proteins used in transport, storage, cell structure, muscular contraction, defense (antibodies, clotting factors, and toxins), and cellular regulation (hormones, effectors, and repressors) are still to be examined by HPLC. Cytosolic proteins, membrane proteins, and the various classes of peptides represent the extremes with which one must deal in polypeptide separations. It is not anticipated that HPLC of these other proteins will present unique chromatographic problems— with the possible exception of nuclear proteins. These proteins are very positively charged and exhibit intermolecular association. However, recent successful RPC separations of ribosomal proteins suggest that the problem can be solved (18). Polynucleotides
The recent expansion of interest in molecular genetics, genetic engineering, and polynucleotide synthesis has prompted a corresponding increase of activity in polynucleotide separations. Although the number of studies on polynucleotide separations by HPLC is small, initial reports are encouraging.
Oligonucleotides. Polynucleotides consisting of less than 50 bases will be referred to here as oligonucleotides. Current interest in this class of com pounds centers on the purification of synthetic oligomers for use in genetic engineering. Oligonucleotides have been separated by RPC, paired-ion RPC, and IEC. In the case of RPC, it has been shown that analytical octadecyl bonded-phase columns can re solve both derivatized and underivatized oligomers up to undecanucleotides with a loading capacity of at least 1 mg (19). Retention is in fluenced by the chemical nature of the purine or pyrimidine bases, the pres ence of hydrophobic protecting groups, the presence of phosphate groups, and oligomer chain length. For example, oligodeoxythymidylates are much more strongly retained than oligodeoxyadenylates of the same chain length (19). Paired-ion RPC is achieved with a positively charged alkylammonium ion acting as the pairing agent for the negatively charged oligonucleotides (20). The technique is capable of re solving oligonucleotides in a homolo gous series of up to 16 bases. However, separations are not strictly on the basis of charge: The octomer of polydeoxyadenylate is eluted after the decamer of polydeoxythymidylate (19). IEC of oligonucleotides has been achieved on two types of anion ex change supports: bonded-phase silicas and nonporous plastics coated with a quaternary amine. The two types of bonded-phase silica supports most widely used are aminoalkyl silanes (21) and cross-linked polyamines (22). Both will easily resolve oligonucleo tide mixtures of up to 20 bases. By the use of 3-μπι particle size polyamine columns, the upper limit of IEC has now been extended to 50 bases. Al though these ion exchange supports are relatively hydrophilic, use of methanol, formamide, or acetonitrile in the mobile phase precludes hydro phobic interactions with columns. Under these conditions retention is predominantly on the basis of charge with elution order being proportional to chain length. Since retention on IEC and RPC columns is based on dif ferent molecular properties, applica tion of both columns to a purification gives better results than rechromatography on either column. The loading capacity of a 0.4 X 15-cm polyamine column is greater than 10 mg of a heptadecanucleotide. Separation of oligonucleotides of up to 50 bases on the coated plastic sup ports also has been reported (23). This support, often referred to as RPC-5, is prepared by the adsorption of trioctylmethylamine onto the surface of a nonporous, hydrophobic plastic parti
cle. Due to the hydrophobic nature of this ion exchange column there is a question as to whether the separation is on the basis of charge, hydrophobic interaction, or a combination of the two modes. The most troublesome fea ture of the RPC-5 column is that ad sorbed amine is easily leached from the support surface. Mobile-phase ve locities and separation times with the RPC-5 column are also much slower than those achieved on silica-based RPC and IEC columns. Transfer ribonucleic acids (tRNAs). RPC-5 columns have been used in the separation of tRNAs. Elu tion is achieved by an ascending salt gradient, implying that the column is behaving predominantly in an ion ex change mode (24). An interesting high-performance analogue of the RPC-5 column was prepared recently (25) by adsorbing trioctylmethylamine onto the surface of an octadecyl silane support. Elution was achieved with combinations of both ionic strength and pH gradients. Although the elution protocol is what would normally be applied to an ion ex change column, resolution results from both IEC and RPC modes. Hy drophobic interactions seem to be the most important in determining the separation of one tRNA from another (25). In some cases, the column will even resolve tRNA isoacceptors. Comparable separations have also been achieved on strict RPC supports eluted with descending salt gradients and 1% propanol (26). Butyl silane supports are the most useful. The in duced solvophobic effect of the highionic-strength mobile phase allows binding and elution under conditions that maintain the native structure of tRNA. Column selectivity is easily
manipulated by mobile-phase temper ature, ionic strength, and the concen tration and type of organic additives. Wehr has shown that SEC also can be useful in the fractionation of methionyl-tRNA (27). Aminoacylated and nonaminoacylated tRNAs may be sep arated by SEC because their shape differences are sufficiently large. Messenger ribonucleic acids (mRNAs). Only a few reports have appeared on the application of HPLC to the purification of mRNAs. When different types of RNA were loaded onto an RPC column and eluted with acetonitrile, all of the mRNA species eluted in a single peak (28). SEC can also be used for the separation of mRNAs from other RNA species. Nearly quantitative recovery and preservation of biological activity in RNA species may be achieved on a TSK G 4000SW column. Although both of these separations are extreme ly useful, life scientists would still like to be able to fractionate the mRNA fraction rapidly into its various gene product subspecies. DNA and its fragments. SEC columns that have been useful in the fractionation of large proteins (>100 KD) also are useful in the frac tionation of polynucleotides (29). However, the bulk of column fraction ations with DNA and its fragments is achieved on RPC-5 or one of its vari ants. The isolation of DNA fragments, particularly bacterial restriction frag ments, is indispensable in nucleic acid research (30). Restriction fragments generally elute from RPC-5 columns in order of size with yields of 69-70%. Occasionally, fragments may bind more tightly to the RPC-5 column than would be predicted from their size (30). This is probably due to an
Figure 4. Separation of Z1-Z10 Hae III restriction fragments of ΦΧ 174 RF DNA (5 μg) on a 30 X 0.4-cm column of uncoated Kel-F82 Adapted from Reference 31
ANALYTICAL CHEMISTRY, VOL. 55, NO. 13, NOVEMBER 1983 · 1305 A
adenine-thymine-rich run within an otherwise guanine-cytosine-rich fragment. It has been noted above that RPC-5 is prepared by coating a hydrophobic quaternary amine onto a nonporous plastic such as polychlorotrifluoroethylene (polyCTFE). As a consequence of the hydrophobic character of this ion exchange support, separations may be of a mixed mechanism. Uncoated polyCTFE will also function as an RPC support when it is eluted with a gradient of acetonitrile (0-18% v/v) in 0.1 M aqueous triethylammonium acetate (31). Elution according to size can be achieved with fragments ranging up to several thousand base pairs. The resolving power of this column with large polynucleotide fragments is shown in Figure 4. Fragments Zl, Z2, and Z3 are approximately 947, 755, and 610 KD, respectively. The Future The future of HPLC in biopolymer fractionation is bright both for theoretical and practical reasons. As theory predicts, the resolving power of microparticulate columns is superior to the larger-particle, gel-type packings in all separation modes. Life scientists will be forced to switch to microparticulate supports to keep up with those who have already made the transition. The fact that separations can also be achieved more quickly with microparticulate supports is a pleasant bonus. New developments will occur in both column technology and applications. In the area of column development, there is a large amount of research being carried out by column manufacturers on totally organic packings because organic materials will have better stability with basic mobile phases. (It should be noted that organic supports probably have no inherent advantage in resolution.) A superior support for the separation of biological macromolecules must be available in a variety of particle and pore sizes and surface areas, be of narrow pore distribution, separate in only one mode at a time, and be mechanically stable to at least 100 bar. Synthesizing a new family of supports with these properties is an undertaking of sufficiently large magnitude that silica-based packings will not be quickly displaced even if organic packings are of superior longevity. The matter of cost is also important; a reduction in the cost of columns would make the question of longevity far less important. I also envision the emergence of high-performance hydrophobic interaction chromatography (HIC) as a major technique for the fractionation of enzymes. The supports will be similar in stationary phase and operate in the same manner as those described
on agarose a decade ago. HIC would circumvent the need for a technique as severe as RPC for the fractionation of proteins in a hydrophobic mode and would complement IEC. Protein fractionation in the sequence of IEC to HIC to SEC would eliminate some of the time presently consumed in dialyses between chromatographic steps and the need for organic solvents in fractionating proteins. Circumventing the need for organic solvents in preparative separations is particularly significant from an economic standpoint. Preliminary evidence is that HIC can have the loading capacity, resolution, and recovery of IEC. The enormous amount of attention directed toward genetic engineering obviously will spill over into chromatographic methods for the separation of polynucleotides, polypeptides in cell lysates, and peptide fragments used in peptide mapping. Column development for polynucleotides will probably receive the most attention because polynucleotides are so important biologically and current methods of purification are so labor-intensive and slow. In 1980, we predicted that 70-80% of all biochemists would use either medium- or high-performance liquid chromatography for the fractionation of proteins by the end of the decade (32). The current level of acceptance of the technique by the life sciences community indicates that this prediction will probably be realized. Acknowledgment The author gratefully acknowledges the assistance of Bill Kopaciewicz, Ed Pfannkoch, Jodi Fausnaugh, and Randy Drager in the preparation of this manuscript. This is journal paper number 9565 of the Purdue Agriculture Experiment Station. References (1) Pearson, J.; Pfannkoch, E.; Régnier, F. E. In "Food Constituents and Food Residues: Their Chromatographic Determination"; Lawrence, James F., Ed.; Marcel Dekker: New York, in press. (2) Hearn, M.T.W.; Hancock, W. S. TIBS 1979,4,58. (3) Hearn, M.T.W.; Hancock, W. S. In "Biological-Biomedical Applications of Liquid Chromatography"; G. L. Hawk, Ed.; Marcel Dekker: New York, 1979. (4) Hermodson, M.; Mahoney, W. Methods Enzymol. 1983.9Î, 352. (5) Régnier, F. Ε. Methods Enzymol. 1983, 91, 137. (6) Bindels, J. G.; DeMan, Β. Μ.; Hoenders, H. J. J. Chromatogr. 1982,252, 255 (7) Hearn, M.T.W.; Régnier, F. E.; Wehr, T. Am. Lab. 1982,14, 18. (8) Lowe, C. R.; Dean, P.D.G. "Affinity Chromatography"; Wiley: New York, 1974; p. 57. (9) Ohlson, S.; Hansson, L.; Larsson, P. O.; Mosbach, K. FEBS Lett. 1983, 93, 5. (10) Lowe, C. R.; Glad, M.; Larsson, P. O.; Ohlson, S.; Small, D.A.P.; Atkinson, T.;
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Mosbach, K.J. Chromatogr. 1981,275, 303. (11) Small, D.A.P.; Atkinson, T.; Lowe, C. R. J. Chromatogr. 1981, 216, 175. (12) Sportsman, J. R.; Wilson, G. S. Anal. Chem. 1980,52,2013. (13) Kasche, V.; Buchholz, K.; Galunsky, B. J. Chromatogr. 1981, 216, 169. (14) Walters, R. R. J. Chromatogr. 1982, 249, 19. (15) Heukeshoven, J.; Dernick, R. J. Chromatogr. 1982,252,241. (16) Gerber, G. E.; Anderegg, R. J.; Herlihy, W. C; Gray, C. P.; Beimann, K.; Khorana, H. G. Proc. Natl. Acad. Sci. U.S.A. 1979,76,227. (17) Régnier, F. E. "Receptor Protein Purification"; Alan R. Liss, Inc.: New York, in press. ( 18) Kerlavage, A. R.; Hasan, T.; Cooperman, B. S. J. Biol. Chem., in press. (19) Haupt, W.; Pingoud, A. J. Chromatogr. 1983,260,419. (20) McLaughlin, L. W.; Romaniuk, E. Anal. Biochem. 1982, 124, 37. (21) Gait, J. J.; Matthes, H.W.D.; Singh, M.; Sprost, B. S.; Titmas, R. C. Nucl. Acid Res. 1982, 10, 6243. (22) Pearson, J. D.; Régnier, F. E. J. Chromatogr. 1983,225, 137. (23) Wells, R. D.; Hardies, S. C; Horn, G. T.; Klein, B.; Larson, J. E.; Neuendorf, S. K.; Panayotatos, N.; Patient, R. K.; Seising, E. Methods Enzymol. 1980,65,327. (24) Pearson, R. L.; Weiss, J. F.; Kelmers, A. D. Biochim. Biophys. Acta 1971,228, 770. (25) Bischoff, R.; Graeser, E.; McLaughlin, L. W. J. Chromatogr. 1983,257, 305. (26) Hjerten, S. Adv. Chromatogr. 1981, 19, 111. (27) Wehr, C. T.; Abbott, S. R. J. Chromatogr. 1979, 785, 453. (28) Simonian, M. H.; Capp, M. H. Second Inter. Symp. HPLC of Prot. Pept. and Polynucl., Baltimore, Md., 1982; Paper No. 404. (29) Kato, Y.; Nakamura, K.; Hashimoto, T. Second Inter. Symp. on HPLC of Prot. Pept. and Polynucl., Baltimore, Md., 1982; Paper No. 403. (30) Patient, R. K.; Hardies, S. C; Larson, E.; Inman, R. B.; Maquat, L. E.; Wells, R. D. J. Biol. Chem. 1979, 254, 5548. (31) Usher, D. A. Nucl. Acid Res. 1979, 6, 2289. (32) Régnier, F. Ε.; Gooding, K. M. Anal. Biochem. 1980,103,1.
Fred Régnier received his BS degree from Nebraska State College in 1960 and his PhD degree from Oklahoma State University in 1965. He joined the faculty at Purdue University in 1969, where he is now professor of biochemistry. His current research focuses on the high-performance liquid chromatography of biopolymers.
