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A new method for the isolation of the trypanocidal factor from normal human serum
Acta Tropwa, 46(1989)71-73
71
Elsevier ATP 00005 Short Communication
A new method for the isolation of the trypanocidal factor from normal human serum P. B a r t h Swzss Tropwal Instttute. Basel, Switzerland
(Recewed 9 June 1988, accepted 18 August 1988) Key words' Trypanocldalfactor, new lsolaUonmethod,Human serum
A novel procedure to isolate the trypanocidal factor from normal human serum (NHS) was developed, using four chromatographic steps: one affinity chromatography, then two anion exchange columns, and a final gel filtration. The isolated fraction showed the same lytic activity against Trypanosoma brucei brucei strains as NHS, whereas NHS-resistant trypanosomes of the Trypanosoma brucei rhodesiense subspecies remained unaffected. The factor is a protein complex of high molecular mass (> 1000 kDa) comprised of four peptides which were visualised under reducing conditions using SDS-polyacrylamide gel electrophoresis. No high density lipoproteins were detectable in the active fraction. Capillary stabilates of the human serum sensitive Trypanosoma brucei brucei clone STIB 345-A (kmdly provided by R. Brun, Swiss Tropical Institute, Basel), or of the Trypanosoma brucei rhodestense stock STIB 704-BABA (prepared following four mouse passages after cloning), were used to infect female ICR mice. 72 h after infecuon, blood was taken by cardiac puncture. Trypanosomes were isolated (Brun and Schoenenberger, 1981), resuspended in phosphate-buffered saline-glucose, pH 8.0, and used for the trypanocidal assay. Assays were performed in 14 ml stoppered plastic tubes, using a modified Minimum Essential Medium with Earle's salts (Jenni and Brun, 1982; Brun and Jenni, 1985). Trypanosomes were incubated at a concentration of 5 x 10 6 cells/ml at 37°C in 2 ml medium containing 10% horse serum and 30% NHS or the volume-equivalent serum fraction. Controls were carried out in the presence of 10% horse serum alone. Normal human serum (NHS) used in these experiments was a pool of sera from five healthy volunteers. Albumin was removed from NHS by affinity chromatography on Blue Sepharose CL-6B (Pharmacia) (Travis et al., 1976). The albumin-depleted serum was concentrated by ultrafiltration through a PM-10 membrane (Amicon) and then chromatographed on a Q Sepharose Fast Flow anion exchange column. Fractions were
Correspondence address Dr B. Betschart,SwissTropical InsUtute,Postfach4002, Basel, Switzerland
0001-706X/89/$03 50 © 1989 ElsevmrScmncePubhshers B V (BiomedicalDivision)
72 collected from peaks of absorbance at 280 nm. The fraction which showed trypanoc]dal activity in the trypanosomal lysis assay was further chromatographed on a Mono Q anion exchange column. Again, one peak showing trypanocidal activity was found The trypanocidal factor in this active fraction was finally purified by gel filtration using a Superose 6 column. For all chromatographic procedures, except albumin removal, FPLC equipment (Pharmacia, Uppsala, Sweden) was utilised. Details of these procedures will be published elsewhere. Low density lipoproteins (LDL) and high density lipoproteins (HDL) were isolated from NHS by ultracentrifugal flotation (Lindgren, 1975) and purified by gel filtration on Superose 6. For SDS-polyacrylamlde gel electrophoresls (SDS-PAGE) of serum fractions a method modified from Laemmli (1970) was used. The purification methods used in this work allowed the ]solation of a serum factor demonstrating trypanolytlc action. Neither HDL nor LDL showed any trypanocidal activity. The number of peptides detected (lane D), plus the high molecular mass (> 1000 kDa), estimated by gel filtration, indicate that the trypanocidal factor is comprised of a multi-peptide complex. Typical peptides of HDL (lane C), i.e. apolipoproteins A-I ( ~ 25 kDa), apo A-II and the C peptides (< 10 kDa) (Scanu et al., 1975), which were found in this HDL preparation, could not be detected in the trypanocidal fraction. Moreover, no correlation was found between the LDL pattern (lane B) and our protein complex. These findings are in marked contrast to
[]
94 kD
A
~t
B G D E
~
o w
67 kD
O
e
43 kD
W
B
80 kD
I
20 kD
I
O
14 kD
Q
O
O
o
Fzg 1 Analysisof LDL (lane B), HDL (lane C) and the trypanocldal frachon (lane D) by SDSpolyacrylamldegel electrophoresls under reducingcon&ttons LanesA and E contain low molecular weight markers Coomassleblue staining
73 p r e v i o u s l y r e p o r t e d results (Rifkin, 1978) a n d conflict with the generally a c c e p t e d t h e o r y o f H D L being the t r y p a n o c i d a l f a c t o r in N H S .
Acknowledgements I wish to t h a n k D r . B. B e t s c h a r t a n d Prof. L. Jen n i for critical discussions a n d for s u p p o r t i n g this w o r k .
References Brun, R. and Jennl, L (1985) Cultwatlon of African and South American trypanosomes of medical or vetennary importance Br Meal Bull 41, 122-129 Brun, R and Sehoenenberger, M. (1981) Stimulating effect of citrate and ClS-aconltate on the transformation of Trypanosomabrucetbloodstream forms to procychc forms m vitro Z Parasltenkd 66, 17-24 Jennl, L and Brun, R (1982) A new In vitro test for human serum resistance of Trypanosoma (T) brucet Acta Trop. 39, 281-284 Laemmh, U K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Lmdgren, F T (1975) Preparative ultracentnfugal laboratory procedures and suggestions for hpoprotem analysis In E G Perkins (Ed.), Analysis of Llplds and Llpoprotelns American Oil Chemists Society, Champaign, IL, pp 204-224 Rlfkm, M.R (1978) Identification of the trypanoctdal factor in normal human serum High density hpoprotem Proc. Natl Acad Scl U S.A 75, 3450-3454. Scanu, A M , Edelstein, C and Kelm, P (1975) Serum lipoproteins. In' F W. Putnam (Ed), The Plasma Proteins Structure, Function and Genetic Control, Academic Press, New York, NY, pp. 326-365 Travis, J , Bowen, J , Tewksbury, D., Johnson, D and Pannell, R (1976) IsolaUon of albumin from whole human plasma and fractlonatlon of albumin-depleted plasma Biochem J 157, 301-306
71
Elsevier ATP 00005 Short Communication
A new method for the isolation of the trypanocidal factor from normal human serum P. B a r t h Swzss Tropwal Instttute. Basel, Switzerland
(Recewed 9 June 1988, accepted 18 August 1988) Key words' Trypanocldalfactor, new lsolaUonmethod,Human serum
A novel procedure to isolate the trypanocidal factor from normal human serum (NHS) was developed, using four chromatographic steps: one affinity chromatography, then two anion exchange columns, and a final gel filtration. The isolated fraction showed the same lytic activity against Trypanosoma brucei brucei strains as NHS, whereas NHS-resistant trypanosomes of the Trypanosoma brucei rhodesiense subspecies remained unaffected. The factor is a protein complex of high molecular mass (> 1000 kDa) comprised of four peptides which were visualised under reducing conditions using SDS-polyacrylamide gel electrophoresis. No high density lipoproteins were detectable in the active fraction. Capillary stabilates of the human serum sensitive Trypanosoma brucei brucei clone STIB 345-A (kmdly provided by R. Brun, Swiss Tropical Institute, Basel), or of the Trypanosoma brucei rhodestense stock STIB 704-BABA (prepared following four mouse passages after cloning), were used to infect female ICR mice. 72 h after infecuon, blood was taken by cardiac puncture. Trypanosomes were isolated (Brun and Schoenenberger, 1981), resuspended in phosphate-buffered saline-glucose, pH 8.0, and used for the trypanocidal assay. Assays were performed in 14 ml stoppered plastic tubes, using a modified Minimum Essential Medium with Earle's salts (Jenni and Brun, 1982; Brun and Jenni, 1985). Trypanosomes were incubated at a concentration of 5 x 10 6 cells/ml at 37°C in 2 ml medium containing 10% horse serum and 30% NHS or the volume-equivalent serum fraction. Controls were carried out in the presence of 10% horse serum alone. Normal human serum (NHS) used in these experiments was a pool of sera from five healthy volunteers. Albumin was removed from NHS by affinity chromatography on Blue Sepharose CL-6B (Pharmacia) (Travis et al., 1976). The albumin-depleted serum was concentrated by ultrafiltration through a PM-10 membrane (Amicon) and then chromatographed on a Q Sepharose Fast Flow anion exchange column. Fractions were
Correspondence address Dr B. Betschart,SwissTropical InsUtute,Postfach4002, Basel, Switzerland
0001-706X/89/$03 50 © 1989 ElsevmrScmncePubhshers B V (BiomedicalDivision)
72 collected from peaks of absorbance at 280 nm. The fraction which showed trypanoc]dal activity in the trypanosomal lysis assay was further chromatographed on a Mono Q anion exchange column. Again, one peak showing trypanocidal activity was found The trypanocidal factor in this active fraction was finally purified by gel filtration using a Superose 6 column. For all chromatographic procedures, except albumin removal, FPLC equipment (Pharmacia, Uppsala, Sweden) was utilised. Details of these procedures will be published elsewhere. Low density lipoproteins (LDL) and high density lipoproteins (HDL) were isolated from NHS by ultracentrifugal flotation (Lindgren, 1975) and purified by gel filtration on Superose 6. For SDS-polyacrylamlde gel electrophoresls (SDS-PAGE) of serum fractions a method modified from Laemmli (1970) was used. The purification methods used in this work allowed the ]solation of a serum factor demonstrating trypanolytlc action. Neither HDL nor LDL showed any trypanocidal activity. The number of peptides detected (lane D), plus the high molecular mass (> 1000 kDa), estimated by gel filtration, indicate that the trypanocidal factor is comprised of a multi-peptide complex. Typical peptides of HDL (lane C), i.e. apolipoproteins A-I ( ~ 25 kDa), apo A-II and the C peptides (< 10 kDa) (Scanu et al., 1975), which were found in this HDL preparation, could not be detected in the trypanocidal fraction. Moreover, no correlation was found between the LDL pattern (lane B) and our protein complex. These findings are in marked contrast to
[]
94 kD
A
~t
B G D E
~
o w
67 kD
O
e
43 kD
W
B
80 kD
I
20 kD
I
O
14 kD
Q
O
O
o
Fzg 1 Analysisof LDL (lane B), HDL (lane C) and the trypanocldal frachon (lane D) by SDSpolyacrylamldegel electrophoresls under reducingcon&ttons LanesA and E contain low molecular weight markers Coomassleblue staining
73 p r e v i o u s l y r e p o r t e d results (Rifkin, 1978) a n d conflict with the generally a c c e p t e d t h e o r y o f H D L being the t r y p a n o c i d a l f a c t o r in N H S .
Acknowledgements I wish to t h a n k D r . B. B e t s c h a r t a n d Prof. L. Jen n i for critical discussions a n d for s u p p o r t i n g this w o r k .
References Brun, R. and Jennl, L (1985) Cultwatlon of African and South American trypanosomes of medical or vetennary importance Br Meal Bull 41, 122-129 Brun, R and Sehoenenberger, M. (1981) Stimulating effect of citrate and ClS-aconltate on the transformation of Trypanosomabrucetbloodstream forms to procychc forms m vitro Z Parasltenkd 66, 17-24 Jennl, L and Brun, R (1982) A new In vitro test for human serum resistance of Trypanosoma (T) brucet Acta Trop. 39, 281-284 Laemmh, U K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Lmdgren, F T (1975) Preparative ultracentnfugal laboratory procedures and suggestions for hpoprotem analysis In E G Perkins (Ed.), Analysis of Llplds and Llpoprotelns American Oil Chemists Society, Champaign, IL, pp 204-224 Rlfkm, M.R (1978) Identification of the trypanoctdal factor in normal human serum High density hpoprotem Proc. Natl Acad Scl U S.A 75, 3450-3454. Scanu, A M , Edelstein, C and Kelm, P (1975) Serum lipoproteins. In' F W. Putnam (Ed), The Plasma Proteins Structure, Function and Genetic Control, Academic Press, New York, NY, pp. 326-365 Travis, J , Bowen, J , Tewksbury, D., Johnson, D and Pannell, R (1976) IsolaUon of albumin from whole human plasma and fractlonatlon of albumin-depleted plasma Biochem J 157, 301-306