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SEPARATION OF PROTEIN MIXTURES BY POLYAMPHOLYTES

УДК 541.64+648.744

Показана возможность разделения смеси белков лизоцима и бычьего сывороточного альбумина (БСА) с использованием линейных полиамфолитов (ПА) на основе диметиламиноэтил метакрилата (ДМАЭМ) и метакриловой кислоты (МК) различного состава: [ДМАЭМ]:[МК] = 38:62 мол % (ПА1) и [ДМАЭМ]: [МК] = 62:38 мол % (ПА2) за счет комплексообразования одного из белков с полиамфолитом.

Сызықты полиамфолит (ПА) көмегімен ақуыздар біреуі есебінен, әр түрлі құрамдағы: метакрилат диметиламиноэтилі (ДМАЭМ) және метакрил қышқылы (МК): [ДМАЭМ]: [МК] = 38:62 мол% (ПА1) және [ДМАЭМ]: [МК] = 62:38 мол% (ПА2) негізіндегі полиамфолиттерді қолдана отырып, бұзау іріткілік альбумин (БСА) және лизоцим ақуыздар қоспаларын бөліп алу мүмкіндігі көрсетілген.

ABSTRACT

Possibility of separation of a mixtures of proteins of lysozyme and bovine serum albumin (BSА) using linear polyampholytes (PA) on a basis of dimethylaminoethyl methacrylate (DMAEM) and methacrylic acid (MAA) various composition [DMAEM]: [MAA] =38:62 mol % (PA1) and [DMAEM]: [МAA] = 62:38 mol % (PA2) due to the complexation of one of proteins and polyampholyte is shown.

Keywords: polyampholyte-protein complexes, water-soluble “annealed” polyampholytes, isoelectric points

INTRODUCTION

Studying of polyampholyte-protein complexes (PPC) is important from the biochemical and biotechnological point of view. Complexation of polyampholytes and proteins can be used for separation and purification of latter, immobilization and stabilization of enzymes. [1]. Commonly used methods for separation of proteins are chromatography, precipitation of proteins by inorganic salts, two-phase precipitation of proteins that is based on uniform distribution of proteins between two aqueous polymer phases [2,3]. The present communication considers the behavior of PPC formed between the water-soluble “annealed” polyampholytes and bovine serum albumin and lysozyme.

EXPERIMENTAL

Copolymers of dimethylaminoethyl methacrylate and methacrylic acid (DMAEM-MAA) were synthesized by radical copolymerization. The compositions of polyampholytes (PA) determined by back potentiometric and conductimetric titrations are equal to [DMAEM]:[MAA] = 38:62 mol% (PA-I) and [DMAEM]:[MAA] = 62:38 mol % (PA-II). Intrinsic viscosities of PA-I and PA-II are equal to [η] = 3,7 dL/g in 0,01 N HCl and [η] = 0,68 in 0,1 M KCl, respectively. The isoelectric points (IEP) of polyampholytes found from the viscometric data are equal to 5.0 for PA-I and 8.6 for PA-II. Bovine serum albumin (BSA) and Lysozyme purchased from the Sigma Chemical Co. had isoionic points (IIP) at pHIIP = 4,7 and pHIIP = 11,0. The intrinsic viscosities of BSA and Lysozyme are equal to 0,08 and 0,07 dL/g, respectively. Reagent-grade KCl, NaOH, HCl and solvents were used. Potentiometric and conductimetric titrations were carried out on the pH/conductivity meter “Mettler Toledo MPC 227” (Switzerland). The viscosity of the solutions was measured in an Ubbelohde viscometer. The composition of PPC was expressed as mass ratio of protein to polyampholytes (Mprot : MPA).

RESULTS AND DISCUSSION

Figure1 shows the isoelectric points (pHIEP) of pure polyampholytes PA-I and PA-II and their complexes with BSA and lysozyme. Both the PA-I and complexes with composition of [MBSA]:[MPA-I] = 2:5 and [Mlysozyme]:[MPA-I] = 1:1 have the same pHIEP = 5,0 and precipitate at this point. PA-II and complexes with composition of [MBSA]:[MPA-II] = 3:2 and [MLysozyme]:[MPA-I] = 1:1 have the values of pHIEP that correspond to 8,6; 8,6 and 8,9, respectively. They are soluble at the range of pH = 2-12. The different isoionic points of BSA and lysozyme suggests that in order to bind proteins the charge of polyampholytes should be arranged between pH = 4,7 and 11,0. Therefore the conditions were selected such a manner that lysozyme might interact only with PA-I via Coulombic interactions. As seen from the curve of ηsp/C –pH at pH = 8,4 the PA-I is charged negatively (Figure 1) and interacts with positively charged Lysozyme. In ternary BSA+Lysozyme+PA-I system the precipitation of PPC was observed at pH = 8,4. After centrifugation and separation of the precipitate the intrinsic viscosity of supernatant was equal to [η] = 0,07 dL/g. This value coincides well with the intrinsic viscosity of pure BSA. Electrophoretic measurements also showed that the supernatant contains only BSA. Thus in ternary system consisting of BSA+Lysozyme+PA-I polyampholyte precipitates the Lysozyme. Adjustment of solution pH to the IEP of pure PA-I (pHIEP = 5.0) leads to precipitation of PA-I itself and detachment of Lysozyme (Scheme 1). Stability of soluble protein-PA-II complexes was investigated with respect to temperature change and thermodynamic quality of solvent (0,1 M KCl-ethanol mixture). Increasing of temperature up to 333 K decreases the intrinsic viscosities of both polyampholytes and PPC particles (Figure 2). Such behavior of the system may be connected with compactization of PPC particles due to strengthening of hydrophobic interactions in aqueous solution. In 0,1 M KCl-ethanol mixture the protein-PA-II complexes are soluble up to 50 vol.% of ethanol content. Further increase of ethanol content resulted in precipitation of PPC.

CONCLUSION

The behavior of polyampholyte-protein system in aqueous solution has been studied as a function of pH and temperature. Separation of protein mixtures by water-soluble polyampholytes was demonstrated.

Figure 1. Dependences of ηsp/C on pH for PA-I (1), [BSA]:[PA-I] (2) and [Lysozyme]:[PA-I] (3) complexes

Figure 2. Temperature dependences of the intrinsic viscosity for PA-II (1), [Lysozyme]: [PA-II] (2) and [BSA]:[PA-II] (3) complexes

Scheme 1. Separation of proteins by polyampholytes

REFERENCES

1. Kudaibergenov S.E., Polyampholytes: Synthesis, Characterization and Application. Kluwer Academic/Plenum Publishers, NY. 2002. 220 p.

2. Patrickios C.S., Hertler W.R., Hatton T.A., Biotechnol. Bioeng. 44 (1994) 1031.

3. Patrickios C.S., Sharma L.R., Armes S.P., Billingham NC., Langmuir. 15 (1999) 1613.