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Apr 12, 2018 - 2 Department of Chemistry, Faculty of Medicine, Syrian Private University, Damascus, Syria. [email protected] Abstract.

ISSN: 2456-6438 [email protected]

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SCHOLARS SCITECH RESEARCHORGANIZATION Journal of Progressive Research in Modern Physics and Chemistry www.scischolars.com

High Resolution Chromatography and Sensitive Retention: Optimization of the Experimental Conditions for Proteins Extraction by Preparative HPLC Loai Aljerf1, Nuha Almasri2 1

Department of Basic Sciences, Faculty of Dental Medicine, Damascus University, Damascus, Syria. 1 [email protected] 2 Department of Chemistry, Faculty of Medicine, Syrian Private University, Damascus, Syria. 2 [email protected]

Abstract High performance liquid chromatography (HPLC) is employed with its various elution systems for the fractionation (by isocratic or gradient elution) of peptides and proteins. Lichrosorb Diol (pore size 100 Ǻ) was chosen for normal partition chromatography of proteins. More details about these separations are illustrated in the current research. The chromatographic capacity and its resolution are investigated and the guidelines are widely defined. Reverse-phase (RP) (more suitable with Lichrosorb RP-8) sorbent was partially dissolved during elution with n-propanol (˂ 40 vol. /vol.) and lyophilized during fractionation. An outstanding resolution of these compounds was seen both at pH 4.0 and 7.5 under room temperature and low flow rate at linear gradient of n-propanol. Selective adsorption had been initiated at pH < 4 and peak broadening was observed when salts eliminated from the eluents. It is suggested by the results of this paper, the use of normal phase with Lichrosorb Diol for the isolation of the soluble heterogeneous proteins in extremely elevated concentrations of organic solvents. Consequently, an exceptional resolution, high capacity, and diminutive elution times were verified for peptides and proteins separations. Keywords: High performance liquid chromatography; Gradient elution; Proteins; Peak broadening; Adsorption. 1 Introduction The use of high-performance sorbents for the liquid chromatography of peptides and proteins is a good technique, early examples being the reverse-phase chromatography of the polypeptide antibiotics actinomycin [1, 2] and bacitracin [3]. Similar methods were later used for the chromatography of various peptides [4]. Elution systems were only developed for the fractionation of peptides and proteins with molecular weights above 10,000 [5, 6]. The use of underivatized silica for fractionating peptides and proteins has also been described [7, 8]. Silica-based sorbents suitable for the chromatography of proteins, developed by Anspach [9], include analytical gel permeation columns, as well as high-capacity ion exchangers. The excellent resolving power of high-performance liquid chromatography (HPLC) is mainly advantageous for the isolation of active components that are present in trace amounts in complex mixtures such as cell extracts or culture growth media. For this purpose fluorometric methods were developed for monitoring peptides and proteins in column effluents at the picomole level [10] as well as compatible methods for their fractionation by HPLC [11]. Fluorometric amino acid analysis of peptide hydrolysates in the picomole range has also been developed [12].

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These HPLC techniques have been further developed and used for the fractionation of proteins. A detailed description of this technique is presented here. The parameters affecting capacity and resolution are discussed and guidelines are provided for fractionation of proteins in general.

2 Materials and Methods The high-pressure liquid chromatography system consisted of a gradient mixer (LKB, Hicksville, N. Y.), a high-pressure minipump (Milton Roy, Riviera Beach, Fla.), and a high-pressure sample injection valve (Altex Scientific, Berkeley, Calif.). An automated, fluorescamine-based detection system was implemented for monitoring proteins in the column effluents [8]. Lichrosorb Diol and Lichrosorb RP-8 (both 10 pm, 4.6 x 250 mm) were obtained from Altex Scientific. Water was purified by a system obtained from Hydro Services and Supplies (Durham, N. C.). Pyridine, formic acid, acetic acid, and n-propanol were distilled from ninhydrin. All column buffers contained thiodiglycol (0.01%’ v/v, Pierce Chemical Co., Rockford, III.).

3 Results and Discussion 3.1 Choice of Sorbent Our experiments have shown that Lichrosorb RP-18 is well suited for the fractionation of peptides of molecular weight up to 12,000. However, small proteins such as ribonuclease (M R 14,000) and chymotrypsinogen (MR 25,000) were found to irreversibly bind to this column. Lichrosorb RP-8, which has the shorter octyl groups bound to the silica matrix, was found to be suitable for the fractionation of proteins of molecular weights up to 30,000. These two sorbents have an average pore size of 100 A. Lichrosorb RP-2 gave poor resolution and low recoveries with proteins. Lichrosorb Diol (pore size 100 Ǻ) was selected for normal partition chromatography of hydrophobic proteins. The various manufacturers use proprietary methods for both production of sorbents and packing of columns. As a result, there are considerable differences in quality and performance. A minimal value of 16,000 theoretical plates per meter for the sorbent with particle size of 10 µm is common. Sorbents of 5-µm particle size have a higher number of theoretical plates, but are more sensitive to pressure fluctuations than are the 10-µm sorbents and therefore require a pulseless solvent delivery system. Analytical-size columns (4.6 x 250 mm) were used in the studies presented below. However, larger columns are commercially available for scaling up these procedures. For preparative applications the chemical stability of the sorbent has to be considered. In one case, a reverse-phase sorbent was partially dissolved during elution and when fractions containing peptides were lyophilized, the silica precipitated and the peptides could not be recovered [7, 13].

3.2 Eluants, pH, and Ionic Strength The eluants that have been used most frequently in normal and reverse-phase HPLC are mixtures of water and methanol or water and acetonitrile. We have introduced n-propanol for reverse-phase HPLC of peptides and proteins. n-Propanol is a powerful hydrophobic eluant, and relatively low concentrations are sufficient for eluting most peptides and proteins tested so far. Thus, previous studies have used gradients from 0 to almost 100% acetonitrile for the fractionation of small peptides on octadecyl silica [14]. In contrast, a gradient from 0 to 20% by volume of n-propanol was found sufficient to elute the same peptides as well as much larger ones from a similar column (Lichrosorb RP- 18) [15]. It was found that the larger the peptide the higher the concentration of organic solvent required for its elution. This is a serious limitation since most of the proteins and large peptides will precipitate from solutions containing high concentrations of such organic solvents. We have successfully applied gradients of n-propanol for the fractionation of proteins on Lichrosorb RP-8. All proteins tested so far were eluted with less than 40% (by volume) n-propanol. Many proteins, including undiluted serum, remain in solution when n-propanol is added to a concentration of 40%. Nevertheless, the solubility of a given protein mixture in 40% n-propanol has to be ascertained prior to reverse-phase chromatography. The pH of the eluant was found to have significant influence on the resolution. Due to the lability of both the proteins and the silica sorbents, the fractionation should be performed in a pH range of 2 to 8. A relatively low resolution was obtained at pH 5-6, probably due to the partial ionization of the carboxyl groups of the proteins. Excellent resolution was observed both at pH 7.5 and 4.0. In addition, Lichrosorb RP-8 displayed different selectivities at pH 7.5 and 4. Therefore, it could be used for a two stage fractionation at these two pH values. It was observed that, similar to amino acids and peptides, higher concentrations of the organic solvent were required for the elution of proteins at the lower pH (Figure 1). The effect of the pH on the fractionation by Lichrosorb Diol (normal phase) was also studied. Good resolution was obtained at pH > 7 only. The role of ionic strength was studied to a limited extent. When salts were omitted from the eluants, peak broadening occurred, probably due to ionic interactions with the sorbents. Therefore, salts (preferably volatile, such as pyridinium formate) were included in the eluants. However, they were washed out with water prior to storage of the columns.

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Fig. 1. Reverse-phase chromatography at pH 7.5 and 4. Ribonuclease (0.5 mg) was chromatographed on a Lichrosorb RP-8 column (4.6 x 250 mm) by a 90-min linear n-propanol gradient (0 to 40%) at pH 7.5 (1 M sodium acetate) and at pH 4.0 (1 M pyridine formate). Row rate was 0.5 ml/min.

3.3 Temperature and Flow Rate These two factors affect the mass transfer of solutes between the mobile and the stationary phases. The rate of diffusion increases at elevated temperature, resulting in a faster transfer of the solutes between the two phases. Therefore, better resolution is normally observed at elevated temperatures. With many proteins, however, this approach cannot be used due to their lability at elevated temperatures, and sometimes very low temperatures are required in order to preserve tertiary structure and biological activity. The present study was restricted to room temperature. Flow rate is much easier to manipulate. Proteins are large molecules that diffuse much more slowly than do low molecular weight compounds. With serum proteins, high resolution was obtained at a flow rate of 1 to 1.5 cm/min (Figure 2). In contrast, low molecular weight compounds have been routinely resolved at much higher flow rates (about 12 cm/min). It has been found useful to fractionate a protein mixture by gel filtration prior to preparative reverse-phase chromatography so that the larger proteins can be separated from the smaller ones. Each class can then be eluted at optimal conditions. In spite of the relatively low flow rates used for proteins, the separations obtained by the HPLC columns are much faster than can be attained by any of the classical protein chromatography techniques and are usually completed in 1 to 3 h. A detailed study of the effect of flow rate on the resolution of proteins is in preparation.

Fig. 2. The effect of flow rate on resolution. Fetal calf serum was extensively dialyzed against NaCl (0.1 M), and 0.5 ml was applied to a Lichrosorb RP-8 column (4.6 x 250 mm). The column was preequilibrated with sodium acetate (1 M, pH 7.5) and eluted by an n-propanol gradient (in two stages). The flow rate was: left, 0.25 ml/min; right, 0.50 ml/min.

3.4 Isocratic vs. Gradient Elution: Capacity and Resolution Proteins can be fractionated either by isocratic or by gradient elution. Isocratic elution is limited to very small sample volumes and thus cannot be readily scaled up. Gradient elution was found more suitable for scale-up. The analytical columns used in this study are normally loaded with up to 2 mg of any material. In order to measure the effect of sample size on the resolution, ribonuclease (0.5, 5.0, and 50 mg, each dissolved in 2 ml of 1 M sodium acetate, pH 7.5) was applied to Lichrosorb RP-8 and the column was eluted by a linear gradient (0 to 40% by volume) of n-propanol at pH

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7.5. It was found that with gradient elution, the high loads had a very small effect on the resolution and thus the analytical column could be used for preparative applications (Figure 3). In another test, the column was saturated with a mixture of proteins and subsequently eluted by the same n-propanol gradient. Although the resolution was reduced, it was still significant due to the high selectivity of the sorbent. Moreover, when the protein mixture was dissolved in a large volume (typically 50 to 100 ml, applied to the column from a reservoir connected to the pump inlet) no loss of resolution was observed in spite of the large volume. This adsorption step eliminated the need for both dialysis and lyophilization.

Fig. 3. Column capacity in chromatography of ribonuclease on Lichrosorb RP-8. Ribonuclease (0.5, 5.0, and 50 mg) was applied to a Lichrosorb RP-8 column (4.6 x 2.50 mm) and eluted by a 3-h linear n-propanol gradient (0 to 40%) in a 1 M pyridine formate buffer (pH 4). The flow rate was 0.25 ml/min, and the fluorometer scale was 1, 10, and 100, respectively.

3.5 Selective Adsorption to the Reverse-Phase Sorbent A further increase in column capacity was attempted by selective adsorption of hydrophobic proteins to Lichrosorb RP-8 in the presence of organic solvents. For instance, n-propanol (0.12 ml) was added to serum (0.38 ml). The mixture was applied to the column and subsequently eluted by a 25 to 40% linear n-propanol gradient. Contrary to the former gradient elution (Figure 2, left), the resolution was completely lost (data not shown). The procedure was then repeated with microgram quantities of dialyzed fetal calf serum. Excellent resolution was obtained for proteins that were eluted by 25% n-propanol; however, proteins that eluted later on appeared as broad peaks (Figure 4). It was therefore concluded that the loss of resolution was due to inefficient binding and not to overloading. The phenomenon was attributed to a dual mechanism of adsorption vs partition. In the absence of an organic solvent adsorption of the proteins to the column in which is rapid and quantitative even at pH 7.5. When an organic solvent is present during application the less hydrophobic proteins partition between the mobile and stationary phases and are eventually washed out. Indeed, the more hydrophobic ones are selectively adsorbed to the support, but in the presence of organic solvents the adsorption is less efficient and thus some of the molecules do not bind to the sorbent at all. Therefore, selective adsorption in the presence of organic solvents at pH ˃ 6 is not practical for most proteins and a preliminary adsorption step in the absence of organic solvents is required for preparative applications. Selective adsorption may be found useful at pH < 4 due to the increased affinity to the sorbent (Figure 1). Isocratic elution, or other modes in which the initial buffer contains an organic solvent, are useful for analytical applications and are applicable to proteins sharing similar affinities to the sorbent.

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Fig. 4. Dual fractionation mechanism on reverse phase column. Dialyzed fetal calf serum (20 µ1) was applied to a Lichrosorb RP-8 column (4.6 x 250 mm) preequilibrated with 25% (v/v) n-propanol-sodium acetate (1 M, pH 7.5). The column was eluted by the same buffer for 18 min and then by a 1-h linear (25 to 40%) n-propanol gradient in the same buffer. The flow rate was 0.25 ml/min.

3.6 Normal-Phase Chromatography of Proteins on Lichrosorb Diol Normal-phase chromatography on Lichrosorb Diol, a novel fractionation technique, was developed for the fractionation of hydrophobic proteins. In several purification procedures, selective precipitation can be obtained by addition of organic solvents. The material which remains soluble can then be fractionated on a normal phase column. For example, npropanol (4 ml) was added to dialyzed calf serum (1 ml), and after centrifugation the supernatant was applied to a Lichrosorb Diol column. The column was then eluted by a gradient of decreasing n-propanol concentration and six peaks were observed (Figure 5). In all cases tested so far, proteins were eluted in the range of 80 to 50% n-propanol. On the other hand, when a mixture of serum (0.6 ml) and n-propanol (0.4 ml) was applied to a Lichrosorb Diol column preequilibrated with n-propanol (50% vol/vol), proteins eluted at the solvent front only without significant fractionation. It was, therefore, concluded that normal-phase chromatography on Lichrosorb Diol can be performed only with proteins that are soluble in very high concentrations of organic solvents (e.g., 80% n-propanol). Other polar-bonded phases, particularly Lichrosorb-NH2, may show up as more general sorbents for normal-phase chromatography of proteins.

Fig. 5. Normal-phase chromatography of proteins on Lichrosorb DioI. Fetal calf serum (1 ml) was extensively dialyzed against NaCl (0.1 M), n-propanol (4 ml) was added, and after centrifugation (10 min, 12,000g) the supernatant was applied to a Lichrosorb Diol column (4.6 x 250 mm). The column was preequilibrated with 80% (v/v) n-propanol-sodium acetate (0.1 M). After elution of unbound proteins, the column was eluted by a 2-h linear gradient from 80 to 50% n-propanol. The flow rate was 0.25 ml/min. Lichrosorb Diol was also used to further fractionate selected peaks obtained by reverse-phase chromatography. In some cases the addition of organic solvents to a protein mixture resulted in the coprecipitation of the hydrophobic proteins with the bulk of the precipitate. However, after fractionation by reverse-phase chromatography, fractions containing hydrophobic proteins were completely soluble in 80% n-propanol and could be further resolved by normal-phase chromatography. Ten to 20 mg of a protein mixture was routinely resolved on the Lichrosorb Diol column.

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3.7 Recovery In the cases tested so far, recoveries were in the range of 50 to 100% at the various stages of purification. This, however, will not always be true, and optimal conditions must be worked out for each protein.

3.8 Guidelines for Protein Fractionation by HPLC The guidelines given below are general and should be optimized in each case. The initial stages in a multistage purification scheme are the classical fractionations by ammonium sulfate or organic solvents. These steps are useful because of the relatively large volumes processed. A volume reduction (dialysis followed by lyophilization, ultrafiltration, etc.) followed by gel permeation chromatography should be performed prior to HPLC. Fractions containing the desired protein are then tested for solubility in 40% n-propanol. If they are soluble, reverse-phase chromatography at pH 7.5 can be attempted. Thus, the fractions obtained from the gel permeation step are adsorbed on the reverse-phase column. Large volumes can be applied to the column from a reservoir connected to the pump inlet thus eliminating a preconcentration step. Next, a steep, n-propanol gradient is applied to bring the organic solvent concentration to about 7- 10% below the elution position of the protein of interest. A shallow gradient (approximately 7 to 15% per hour) is subsequently applied for maximal resolution at the desired region (see Figure 2, left). Fractions soluble in 80% n-propanol can then be further fractionated by normal phase chromatography on Lichrosorb Diol at pH 7.5 and subsequently by reverse phase chromatography at pH 4. The Lichrosorb Diol step should be omitted if the fraction of interest is insoluble in 80% n-propanol. In one case, a combination of such procedures led to severalthousand fold purification.

4 Conclusion Novel preparative methods for the fractionation of proteins have been developed. Excellent resolution, high capacity, and short elution times were demonstrated. These methods are not applicable to proteins that are irreversibly denatured by organic solvents. Nevertheless, the powerful resolution makes them some of the best preparative techniques available for the fractionation of heterogeneous protein mixtures that are encountered in many biochemical studies. Similar applications were previously performed for the isolation and purification of human leukocyte interferon [6,8]. Proopiocortin, the common precursor to corticotropins and endorphin [16] was subsequently purified.

References [1] Martin, D. G., Peltonen, R. E., Nielsen, J. W. (1986). Preparative resolution of an actinomycin complex by countercurrent chromatography in the Ito coil planet centrifuge. J. Antibiot., 39(5), 721-723. https://doi.org/10.7164/antibiotics.39.721. [2] Minshull, T. C., Cole, J., Dockrell, D. H., Read, R. C., Dickman, M. J. (2016). Analysis of histone post translational modifications in primary monocyte derived macrophages using reverse phase×reverse phase chromatography in conjunction with porous graphitic carbon stationary phase. J. Chromatogr. A., 1453, 43-53. https://doi.org/10.1016/j.chroma.2016.05.025. [3] Potts, A. R., Psurek, T., Jones, C., Parris, L., Wise, A. (2012). Validation of a quantitative HPLC method for bacitracin and bacitracin zinc using EDTA as a mobile-phase modifier. J. Pharm. Biomed. Anal., 70, 619-623. https://doi.org/10.1016/j.jpba.2012.06.016. [4] Alwera, S., Bhushan, R. (2017). RP-HPLC enantioseparation of β-adrenolytics using micellar mobile phase without organic solvents. Biomed. Chromatogr., 31(11), e3983. https://doi.org/10.1002/bmc.3983. [5] Yphantis, D. A. (2006). Rapid determination of molecular weights of peptides and proteins. Ann. N. Y. Acad. Sci., 88(3), 586-601. https://doi.org/10.1111/j.1749-6632.1960.tb20055.x. [6] Aljerf, L., Choukaife, A. E. (2017). A novel method to chromatographically resolution of sulphonamides by vapour-programmed Thin-Layer Chromatography. MOJ Bioorg. Org. Chem., 1(4), 00024. https://doi.org/10.15406/mojboc.2017.01.00024. [7] Meyerson, L. R., Abraham, K. I. (1986). High performance liquid chromatographic properties of peptides and proteins on a dihydroxyalkyl bonded silica stationary phase. Peptides., 7(3), 481-489. https://doi.org/10.1016/0196-9781(86)90018-5. [8] Aljerf, L., Alhaffar, I. (2017). Salivary distinctiveness and modifications in males with Diabetes and Behçet's disease. Biochem. Res. Int., 2017, 1-12. https://doi.org/10.1155/2017/9596202. [9] Anspach, F. B. (1994). Silica-based metal chelate affinity sorbents II. Adsorption and elution behavior of proteins on iminodiacetic acid affinity sorbents prepared via different immobilization techniques. J. Chromatogr. A., 676(2), 249-266. https://doi.org/10.1016/0021-9673(94)80425-7.

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[10] Říčný, J., Tuček, S., Vinš, I. (1992). Sensitive method for HPLC determination of acetylcholine, choline and their analogues using fluorometric detection. J. Neurosci. Methods., 41(1), 11-17. https://doi.org/10.1016/0165-0270(92)90119-x. [11] Johnsen, A. A. (1991). Nondestructive amino acid analysis at the picomole level of prolinecontaining peptides using aminopeptidase M and prolidase: Application to peptides containing tyrosine sulfate. Anal. Biochem., 197(1), 182-186. https://doi.org/10.1016/0003-2697(91)90376-5. [12] Kondo, N., Imai, K., Isobe, M., Goto, T. (1984). A low picomole fluorometric detection system for amino acid analysis. Agric. Biol. Chem., 48(6), 1595-1601. https://doi.org/10.1271/bbb1961.48.1595. [13] Sasaki, Y., Coy, D. H. (1987). Solid phase synthesis of peptides containing the CH2NH peptide bond isostere. Peptides., 8(1), 119-121. https://doi.org/10.1016/0196-9781(87)90174-4. [14] Prokopov, S. V., Kurbatova, S. V., Davankov, V. A., Il’in, M. A. (2012). Chromatographic retention of adamantylamidrazones and triazoles by octadecyl silica gel and hypercrosslinked polystyrenes from wateracetonitrile solutions. Russ. J. Phys. Chem. A., 86(5), 852-859. https://doi.org/10.1134/s0036024412050299. [15] Kalchenko, O. I., Cherenok, S. O., Savonik, L. I., Solovyov, A. V., Gorbachuk, V. V., Kalchenko, V. I. (2014). Investigation of sorption of calix[4]arene and calix[4]resorcinarene tetraalkyl derivatives with the LiChrosorb RP 18 surface by RP HPLC and molecular modelling methods. Ž. org. farm. hìm., 12(3), 17-22. https://doi.org/10.24959/ophcj.14.799. [16] Naito, N., Takahashi, A., Nakai, Y., Kawauchi, H. (1984). Immunocytochemical identification of the proopiocortin-producing cells in the chum salmon pituitary with aantisera to endorphin and NH2terminal peptide of salmon proopiocortin. Gen. Comp. Endocrinol., 56(2), 185-192. https://doi.org/10.1016/0016-6480(84)90029-7.

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