RP-HPLC를 이용한 혈액에 포함된 펩타이드 분리

이승기, Yulia Polyakova, 노경호

초정밀생물분리기술센터, 인하대학교, 화학공학과

Isolation of Peptides from Human Blood by RP-HPLC

Theories and Applications of Chem. Eng., 2002, Vol. 8, No. 1

Sueng Ki Lee, Yulia Polyakova and Kyung Ho Row

Center for Advanced Bioseparation Technology and Dept. of Chem. Eng.,

Inha University, 253 Yonghyun-Dong, Nam-Ku, Incheon 402-751, Korea

Introduction

The processes of separation of biological-active substances are very important task in modern chemistry, biology, biochemistry and medicine. Now there is information about the peptides, which has wide spectrum of biological activity. Researches on these peptides become interesting and urgent. However, the formation of such peptides in human blood has been investigated very insufficiently.[1] Thus, a study on the separation of the peptides by HPLC  will surely lead to the basis for the various types of research.

Reversed-phase high-performance liquid chromatography (RP-HPLC) is the most widely used analytical technique for separation and isolation of peptides.[2] Peptide isolation in RP-HPLC mainly depends on its amino acid composition, but also on peptide chain length and sequence, as well as on the corresponding stationary phase and chromatographic condition for elution.[3-5] So the mobile phase is a mixture of an aqueous and organic solvent in which the hydrophobic interaction between peptides and the non-polar stationary phase allows peptide isolation. Peptide elution from the column occurs by increasing the percentage of the organic modifier. The mobile phase is essentially fixed since most isolations are conducted with shallow gradients using aqueous buffers modified with acetonitrile. Acetonitrile is generally employed owing to its UV transparency, low viscosity and volatility. It also provides greater resolution and selectivity being the best solvent for most practical purposes.[3] The mobile phase for peptide chromatography contains an ionic modifier which acts as counter-ion of any charged amino acids. This ionic modifier increases peptide hydrophobicity by forming ion-pair complexes. Trifluoroacetic acid(TFA) has been commonly used as an ion pair-forming reagent for peptide isolation. TFA is an excellent polypeptide solvent, is an strong ion-pairing reagent, is transparent to ultraviolet light, and is easily eliminated by lyophilization.[3] Silica pore-size is selected depending on peptide size. Very large peptides and proteins 300Å, whereas lower pore size (<125Å) is suitable for small peptides. In this work, the peptides were separated with the pore size of 100Å.

The purpose of this work was to isolate of the peptides, by reverse-phased high performance liquid chromatography with UV detector.

화학공학의 이론과 응용 제8권 제1호 2002년

Experimental

1. Reagents and standard peptides

Most standard peptides were commercially obtained from Sigma (St Louis, MO, USA). HPLC grade solvent, acetonitrile was from J. T. Baker (Philipsburg NJ, USA). Trifluoroacetic Acid (TFA) was purchased from Sigma (St. Louis, MO, USA). Water filtered by Milipore ultra-pure water system (Milipore, Bedford, MA, USA).

2. Sample preparation

The sample was prepared in High-Purity Separation Lab., Inha University. This sample make by several steps.

(1) Take a fresh sample of blood, 9ml, and it was dissolved in 1 ml of sodium citrate solution.

(2) Centrifuge the blood (10min, 602g=2280rpm)

(3) Throw away the cell element of plasma in upper side.

(4) Add 0.154M NaCl solution into the part in the lower side with pH (=7.4).

(5) Repeat 3 times of the procedures, (2) ~ (4).

(6) Collect the fraction of erythrocytes in bottom side as shown in Fig.1.

(7) Add 15% trichloroaceticacids(TCA) solution to fraction of erythrocytes.

(0.5 ml of erythrocytes into 1.0ml of TCA)

(8) Centrifuge the sample of (7) (10min, 1000g=3120rpm)

(9) Analyze a liquid above a deposit. See Fig.2.

The constant injection volume of extract solution, 5□, was used throughout.

3. Apparatus and method

HPLC was performed with Waters 600S solvent delivery system and 2487 UV dual channel detector (Waters, Milford, MA, U.S.A.). Data acquisition system was Millennium 3.2 installed in HP Vectra 500 PC. Dual UV wavelengths were set at 254nm and 220nm. The mobile phases were degassed with helium. The flow rate of mobile phase was fixed at 1.0 ml/min.

The mobile phases were acetonitrile, water respectively. Gradient mode of mobile phase was applied to adjust the total run times between 20 and 30 min. The column was purchased from Alltech Co. The column size was 0.46×15 cm and packed by C18, 100Å, 5□. All the experimental runs were carried out in ambient temperature.

Results and Discussion

The gradient-elution mode was employed to isolate peptides. It is very difficult to resolve peptides in isocratic mode, because peptides have polarity which are different with each other. In addition, several gradient steps were necessitated, and the five linear-gradient steps were utilized in this work. To adjust the experimental conditions, the modifier composition of TFA was changed from 0.1 to 0.75 vol. %. The complete mobile phase condition was listed in Table 1. The resulting chromatogram was shown in Fig. 3.

It is expected that more concrete understanding of peptide-isolation will provide very useful information to the function of peptides in human blood. All the peaks in Fig.3 will be identified by standard chemicals and LC-MS.

Table 1. Experimental conditions in Fig.3

Gradient time (min)	0.75% TFA in water vol.%	Acetonitrile vol.%
Initial	100	0
4	90	10
7	80	20
10	65	35
20	50	50
30	0	100

Acknowledgements

The authors gratefully acknowledged for the financial support by Center for Advanced Bioseparation Technology.

Theories and Applications of Chem. Eng., 2002, Vol. 8, No. 1

Reference

1. Ivanov V.T., Karelin A.A., Philippova M.M., Nazimov I.V., Pletnev V.Z., Biopolymers.     Peptide Science., (1997). V. 43. P. 171-188.1.

2. V. Casal, P.J. Martin-Alvarez, T. Herraiz, Analytica Chimica Acta., 326 (1996) P. 77-84

3. T. Herraiz., Analytica Chimica Acta., 352 (1997) P. 119-139

4. M.C.J Wilce, M.I. Aguilar and M.T.W. Hearn, J. Chromatogr., 536 (1991) P. 165

5. C.T. Mant, T.W.L. Burke, J.A. Black and R.S. Hodges, J. Chromatogr., 458 (1998) P. 193

Fig.1 Photograph showing leukocytes in the upper side and erythrocytes in the lower side.

Fig.2 Photograph showing peptides in the upper side and erythrocytes in the lower side.

Fig.3 Chromatogram of isolated peptides (eluent A: 0.75% TFA     in water, eluent B: acetonitrile. Flow rate: 1ml/min. Detection      by absorbance at 254nm)

화학공학의 이론과 응용 제8권 제1호 2002년