Comp Biochem Phvstol
1976 l'ol 54B pp 141 to 143 Pergamon Press Printed In Great Britain
MYOGLOBIN FROM THE CILIATE PROTOZOAN PARAMECIUM AURELIA R H DAVIS, JR AND E STEERS,JR Laboratory of Chemical Biology, NIAMDD, NIH, Bethesda, MD 20014, U S A (Recewed 23 Aprd 1975) Abstract--1 The respiratory pigment ~solated from cell extracts of the protozoan Paramecium aureha (referred to as myoglobm) was partmlly purified and resolved into five electrophoretlcally &stmct components (Mbl-Mb5) 2 The separated components appear to have identical mol wt of approx 16,000 3 Isoelectnc focusing resulted m isoelectnc points of 5 54, 5 18, 498, 4 51 and 4 16 respectively 4 The five forms represent 32, 11, 12, 33 and 12~o of the total haemoprotem materml respectwely
buffer, pH 7 2 which contained 0 0270 KCN The eluate was measured spectrophotometncally at 280 and 416 nm The appropriate fractions were pooled and precipitated w~th sohd ammonium sulfate to a final concentration of 6570 and stored at 4°C The partially purified material was dmlyzed agamst phosphate buffer for 24 hr prior to electrophoresis Ahquots of 1502 containing between 200 and 250/lg of haemoprotem (van Assendelft, 1970) were electrophoresed on 757o standard polyacrylamlde &sc gels
INTRODUCTION
The presence of a "haemoglobm-hke" pigment in the clhated protozoan Paramecmm was first reported by Sato & Tamlya (1937) and subsequently by Keihn & Hartree (1953) on the basis of spectroscopic observations on whole cell suspensions More recently Smith et al (1962) described, m a prehmmary study, observations on partially purified preparations from cell homogenates These investigators described the spectral properties for several denvatwes of a respiratory pigment which reversibly brads oxygen and estimated its mol wt based on sedmaentatlon and diffusion measurements They concluded that the protein has a mol wt of 13,000 and is monomerlc with respect to the heme This paper reports the presence of five electrophoret~cally distinct components of the haem-contammg protein of Paramecium aureha which appears to have ldenhcal mol wt of 16,000 The lsoelectrlc pomts of the separate components ranged from 5 54 to 4 16 The five electrophoretically different species consisted of two major forms comprising 65~o of the total and three minor forms which account for the remaining 35~o The relative concentrations of the respective bands remained consistent from culture to culture
A
B
C
tt Mbl
Mb2
';
MATERIALS AND METHODS
Cultures were grown from single cell isolations on a grass infusion or Cerophyl (Cerophyl Laboratories Inc, Kansas Cxty, Mo ) buffered with Na2HPO4 7H20 and expanded to a total of 961 by a procedure previously described (Pollack & Steers, 1973) The animals were harvested at maximum stationary phase by low speed centrlfugatlon using a modified cream separator (Preer & Preer, 1959) The concentrated cell suspensmn was homogemzed m if2 M raffinose, 005 M phosphate buffer, pH 7 2, ffl M NaC1 at 4°C The cell debris was removed centrifugally and the supernatant fractlonated with saturated ammomum sulfate The preclpltate obtamed between 50 and 6570 saturation was retained Th~s precxp~tated fraction was dissolved in 270 K3Fe(CN)6, 0 570 KCN, 0 05 M phosphate buffer, pH 7 2 to convert the haemoprotem to the more stable cyanmet form (van Assendelft, 1970) The sample was chromatographed on a column (2 x 75cm) of Sephadex G-75 and was eluted with 005M phosphate 141
Mb4 It
Mb5
Fig 1 Electrophoresis of partially purified Paramecmm myoglobm run on standard 7 5~ polyacrylamlde disc gels (A) Unstained disc gel showing posmon and mtensltles of red pigment (B) Gel stained specifically for haem with benzldene HCI (C) Gel stained for total protein with 0 57o amldo-Schwartz in 7 5% acetic acid The disc samples contamed approx 200-250/ag of haemoprotem (van Assendelft, 1970)
C
Fig 2 Electrophoresls of partially purified Paramecium myoglobin run on 5°o polyacrylamlde disc gels containing 0 1% Na dodecyl sulfate according to the procedure of Shapiro et al (1967) (A) Unstained gel (B) Gel stained specifically for haem with benzldlne HCI (C) Gel stained for total protein with 0 5°0 amldo Schwartz in 7 5% acetic acid The disc samples contained approx 20~250 fig of haemoproteln (van Assendelft, 1970)
M
B
i
~J~
:3
4
/
/
I pH
5
/"
/
,,o
I 6
7
-4
-
-2
5
3
Fig 3 Isoelectrlc focusing of Paramecium myoglobln in a pH gradient using pH 4~6 ampholytes in 5°o polyacrylamlde gels according to the procedure described by Fawcett (1968) Approximately 2 mm slices were cut from select positions of the gel, incubated in 1 ml of distilled H20 and measured for pH Solid circles represent pigmented bands, open circles represent remaining areas of gel Identical experiments were carried out in a pH gradient using pH 3 5~10 ampholytes with similar results
Mb5
Mb4
Mb3
Mb2
Mbl
Z (7
> .
1976 l'ol 54B pp 141 to 143 Pergamon Press Printed In Great Britain
MYOGLOBIN FROM THE CILIATE PROTOZOAN PARAMECIUM AURELIA R H DAVIS, JR AND E STEERS,JR Laboratory of Chemical Biology, NIAMDD, NIH, Bethesda, MD 20014, U S A (Recewed 23 Aprd 1975) Abstract--1 The respiratory pigment ~solated from cell extracts of the protozoan Paramecium aureha (referred to as myoglobm) was partmlly purified and resolved into five electrophoretlcally &stmct components (Mbl-Mb5) 2 The separated components appear to have identical mol wt of approx 16,000 3 Isoelectnc focusing resulted m isoelectnc points of 5 54, 5 18, 498, 4 51 and 4 16 respectively 4 The five forms represent 32, 11, 12, 33 and 12~o of the total haemoprotem materml respectwely
buffer, pH 7 2 which contained 0 0270 KCN The eluate was measured spectrophotometncally at 280 and 416 nm The appropriate fractions were pooled and precipitated w~th sohd ammonium sulfate to a final concentration of 6570 and stored at 4°C The partially purified material was dmlyzed agamst phosphate buffer for 24 hr prior to electrophoresis Ahquots of 1502 containing between 200 and 250/lg of haemoprotem (van Assendelft, 1970) were electrophoresed on 757o standard polyacrylamlde &sc gels
INTRODUCTION
The presence of a "haemoglobm-hke" pigment in the clhated protozoan Paramecmm was first reported by Sato & Tamlya (1937) and subsequently by Keihn & Hartree (1953) on the basis of spectroscopic observations on whole cell suspensions More recently Smith et al (1962) described, m a prehmmary study, observations on partially purified preparations from cell homogenates These investigators described the spectral properties for several denvatwes of a respiratory pigment which reversibly brads oxygen and estimated its mol wt based on sedmaentatlon and diffusion measurements They concluded that the protein has a mol wt of 13,000 and is monomerlc with respect to the heme This paper reports the presence of five electrophoret~cally distinct components of the haem-contammg protein of Paramecium aureha which appears to have ldenhcal mol wt of 16,000 The lsoelectrlc pomts of the separate components ranged from 5 54 to 4 16 The five electrophoretically different species consisted of two major forms comprising 65~o of the total and three minor forms which account for the remaining 35~o The relative concentrations of the respective bands remained consistent from culture to culture
A
B
C
tt Mbl
Mb2
';
MATERIALS AND METHODS
Cultures were grown from single cell isolations on a grass infusion or Cerophyl (Cerophyl Laboratories Inc, Kansas Cxty, Mo ) buffered with Na2HPO4 7H20 and expanded to a total of 961 by a procedure previously described (Pollack & Steers, 1973) The animals were harvested at maximum stationary phase by low speed centrlfugatlon using a modified cream separator (Preer & Preer, 1959) The concentrated cell suspensmn was homogemzed m if2 M raffinose, 005 M phosphate buffer, pH 7 2, ffl M NaC1 at 4°C The cell debris was removed centrifugally and the supernatant fractlonated with saturated ammomum sulfate The preclpltate obtamed between 50 and 6570 saturation was retained Th~s precxp~tated fraction was dissolved in 270 K3Fe(CN)6, 0 570 KCN, 0 05 M phosphate buffer, pH 7 2 to convert the haemoprotem to the more stable cyanmet form (van Assendelft, 1970) The sample was chromatographed on a column (2 x 75cm) of Sephadex G-75 and was eluted with 005M phosphate 141
Mb4 It
Mb5
Fig 1 Electrophoresis of partially purified Paramecmm myoglobm run on standard 7 5~ polyacrylamlde disc gels (A) Unstained disc gel showing posmon and mtensltles of red pigment (B) Gel stained specifically for haem with benzldene HCI (C) Gel stained for total protein with 0 57o amldo-Schwartz in 7 5% acetic acid The disc samples contamed approx 200-250/ag of haemoprotem (van Assendelft, 1970)
C
Fig 2 Electrophoresls of partially purified Paramecium myoglobin run on 5°o polyacrylamlde disc gels containing 0 1% Na dodecyl sulfate according to the procedure of Shapiro et al (1967) (A) Unstained gel (B) Gel stained specifically for haem with benzldlne HCI (C) Gel stained for total protein with 0 5°0 amldo Schwartz in 7 5% acetic acid The disc samples contained approx 20~250 fig of haemoproteln (van Assendelft, 1970)
M
B
i
~J~
:3
4
/
/
I pH
5
/"
/
,,o
I 6
7
-4
-
-2
5
3
Fig 3 Isoelectrlc focusing of Paramecium myoglobln in a pH gradient using pH 4~6 ampholytes in 5°o polyacrylamlde gels according to the procedure described by Fawcett (1968) Approximately 2 mm slices were cut from select positions of the gel, incubated in 1 ml of distilled H20 and measured for pH Solid circles represent pigmented bands, open circles represent remaining areas of gel Identical experiments were carried out in a pH gradient using pH 3 5~10 ampholytes with similar results
Mb5
Mb4
Mb3
Mb2
Mbl
Z (7
> .
