ANALYTICAL
BIOCHEMISTRY
The Determination
90,
447-450
(1978)
of Myoglobin
by Gel Chromatography
The method recently described for the determination of haemoglobin in ovine muscles (1) has now been modified for the measurement of myoglobin. Evidence is presented here for the validity of using it for myoglobin determination in muscles of the rat, pig, sheep, and ox. MATERIALS
AND METHODS
Chemicals were Analar quality from BDH Chemicals, Ltd., Poole, England. Hibitane was obtained from ICI, Ltd., Macclesfield, England. Muscles were removed postrigor and were cleaned of any external fat and connective tissue. Samples of 3-4 g were homogenised for 20 s with 20 ml of ice-cold 0.04 M phosphate buffer at pH 6.8 using a Silverson laboratory homogeniser with microtubular head (Silverson Machines, Ltd., Chesham, England). The homogenate, after storage for 1 h at 4°C. was centrifuged at 65OOg for 10 min to remove gross cell debris and a few micrograms of sodium cyanide and sodium nitrite were then added to convert the pigments in the supematant to the cyanmet forms. The extract was cleared by centrifugation at 30,OOOg and 15°C for 60 min and solid potassium chloride was added to bring the concentration to about 0.4 M. Between 100 and 200 ~1 of this extract containing 5-80 pg of myoglobin was chromatographed on Sephadex G-50, as described previously (l), except that the column was 0.6 x 75 cm and the buffer contained 0.002% Hibitane. The flow cell had a reduced volume of 12 ~1 and a path length of 1.5 cm in order to increase the sensitivity and resolution of the system. Buffer was pumped through the system at 8 ml/h with a chart speed of 6 cm/h. The effluent was monitored at 420 nm using a CE 303 spectrophotometer (Cecil Instruments, Ltd., Cambridge, England). An elution profile is shown in Fig. 1. Identity of the pigment in each peak was checked in initial experiments by their specific absorption spectra, after they were collected in separate fractions. Peak heights were converted to absorbance units by the method described by Coulson (2) and the quantity of myoglobin in the chromatographed sample was determined by comparison of the peak heights with those of standards. Pigment concentration in the muscle was calculated by taking the water content of muscle as 75% of its wet weight. Recovery of added myogfobin. Purified myoglobin from rat, pig, sheep, and ox muscle was added to muscle samples and these were analysed as described above. With rat muscles, the muscle of one side had myoglobin added to it, and the contralateral muscle acted as the control. The pig, 447
0003-2697/78/0901-0447$02.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
448
SHORT
COMMUNICATIONS
SHORT
449
COMMUNICATIONS
sheep, and ox muscle contained initially 1.83, 2.53, and 5.06 mg of myoglobin/g of muscle, respectively. The myoglobin concentration in the rat muscles used ranged from 1.2 1 to 2.67 mgig. Mean (? SD) recoveries were 100.1 t 4.0% for ox (n = 4), 100.5 ? 3.5% for sheep (n = 5), 100.1 -+ 2.1% for pig (n = 4), and 101.7 * 3.2% for rat (n = 6). DISCUSSION
It was found that peak height gave a reliable measure of myoglobin concentration, the sensitivity being about 0.009 absorbance units/pg of myoglobin. This enabled 2 pg of myoglobin to be detected and 5 pg to be measured. The use of myoglobin standards, rather than measurement of total pigment concentration from absorbance at 540 nm and calculation of the percentage myoglobin in the extract from the chromatogram (l), was necessary because of the presence in some extracts of significant amounts of an interfering pigment. This was eluted just before the haemoglobin and was frequently present in rat and pig muscle extracts, but very rarely in those from sheep or ox. The pigment was also difficult to quantify accurately from the chromatogram due to the variable peak shape. It appeared to correspond to hemalbumin as found by Akesonet al. (3) in human muscle and is seen in the second pig sample in Fig. 1. Consecutive samples overlap slightly in the chromatogram in Fig. 1, the absorbance not quite returning to the baseline which is, therefore, obtained from that at the beginning and end of the recording. Small fluc-
Myoglobin
FIG.
2. Peak
height
plotted
against
quantity
( w)
of myoglobin
for the standards
in Fig.
1.
450
SHORT COMMUNICATIONS
tuations in the elution profiles are probably mainly due to changes in refractive index of the eluant caused by addition of potassium chloride to the samples, also due to some extent to the slight absorbance by interfering proteins in the extracts. Figure 2 shows a linear relationship between peak height and the amount of myoglobin in the standards in Fig. 1, and in routine use one standard was sufficient for each chromatogram. Reproducibility was satisfactory; the standard deviation of the mean of duplicates over 14 samples of bovine muscle was ? 0.125 mg/g for an overall mean myoglobin concentration of 4.08 mg/g. PAUL ARC Meat Research Institute, Bristol BSl8 7 DY. England Received January 31. 1978
D. WARRISS
Lay&d
REFERENCES Warriss, P. (1976) Anal. Biochem. 72, 104. Coulson, A. F. W. (1972) Anal. B&hem. 49, 589. 3. Akeson, A.. Biorck, G.. and Simon, R. (1968) Acta Med. I. 2.
Stand.
183, 307.
BIOCHEMISTRY
The Determination
90,
447-450
(1978)
of Myoglobin
by Gel Chromatography
The method recently described for the determination of haemoglobin in ovine muscles (1) has now been modified for the measurement of myoglobin. Evidence is presented here for the validity of using it for myoglobin determination in muscles of the rat, pig, sheep, and ox. MATERIALS
AND METHODS
Chemicals were Analar quality from BDH Chemicals, Ltd., Poole, England. Hibitane was obtained from ICI, Ltd., Macclesfield, England. Muscles were removed postrigor and were cleaned of any external fat and connective tissue. Samples of 3-4 g were homogenised for 20 s with 20 ml of ice-cold 0.04 M phosphate buffer at pH 6.8 using a Silverson laboratory homogeniser with microtubular head (Silverson Machines, Ltd., Chesham, England). The homogenate, after storage for 1 h at 4°C. was centrifuged at 65OOg for 10 min to remove gross cell debris and a few micrograms of sodium cyanide and sodium nitrite were then added to convert the pigments in the supematant to the cyanmet forms. The extract was cleared by centrifugation at 30,OOOg and 15°C for 60 min and solid potassium chloride was added to bring the concentration to about 0.4 M. Between 100 and 200 ~1 of this extract containing 5-80 pg of myoglobin was chromatographed on Sephadex G-50, as described previously (l), except that the column was 0.6 x 75 cm and the buffer contained 0.002% Hibitane. The flow cell had a reduced volume of 12 ~1 and a path length of 1.5 cm in order to increase the sensitivity and resolution of the system. Buffer was pumped through the system at 8 ml/h with a chart speed of 6 cm/h. The effluent was monitored at 420 nm using a CE 303 spectrophotometer (Cecil Instruments, Ltd., Cambridge, England). An elution profile is shown in Fig. 1. Identity of the pigment in each peak was checked in initial experiments by their specific absorption spectra, after they were collected in separate fractions. Peak heights were converted to absorbance units by the method described by Coulson (2) and the quantity of myoglobin in the chromatographed sample was determined by comparison of the peak heights with those of standards. Pigment concentration in the muscle was calculated by taking the water content of muscle as 75% of its wet weight. Recovery of added myogfobin. Purified myoglobin from rat, pig, sheep, and ox muscle was added to muscle samples and these were analysed as described above. With rat muscles, the muscle of one side had myoglobin added to it, and the contralateral muscle acted as the control. The pig, 447
0003-2697/78/0901-0447$02.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
448
SHORT
COMMUNICATIONS
SHORT
449
COMMUNICATIONS
sheep, and ox muscle contained initially 1.83, 2.53, and 5.06 mg of myoglobin/g of muscle, respectively. The myoglobin concentration in the rat muscles used ranged from 1.2 1 to 2.67 mgig. Mean (? SD) recoveries were 100.1 t 4.0% for ox (n = 4), 100.5 ? 3.5% for sheep (n = 5), 100.1 -+ 2.1% for pig (n = 4), and 101.7 * 3.2% for rat (n = 6). DISCUSSION
It was found that peak height gave a reliable measure of myoglobin concentration, the sensitivity being about 0.009 absorbance units/pg of myoglobin. This enabled 2 pg of myoglobin to be detected and 5 pg to be measured. The use of myoglobin standards, rather than measurement of total pigment concentration from absorbance at 540 nm and calculation of the percentage myoglobin in the extract from the chromatogram (l), was necessary because of the presence in some extracts of significant amounts of an interfering pigment. This was eluted just before the haemoglobin and was frequently present in rat and pig muscle extracts, but very rarely in those from sheep or ox. The pigment was also difficult to quantify accurately from the chromatogram due to the variable peak shape. It appeared to correspond to hemalbumin as found by Akesonet al. (3) in human muscle and is seen in the second pig sample in Fig. 1. Consecutive samples overlap slightly in the chromatogram in Fig. 1, the absorbance not quite returning to the baseline which is, therefore, obtained from that at the beginning and end of the recording. Small fluc-
Myoglobin
FIG.
2. Peak
height
plotted
against
quantity
( w)
of myoglobin
for the standards
in Fig.
1.
450
SHORT COMMUNICATIONS
tuations in the elution profiles are probably mainly due to changes in refractive index of the eluant caused by addition of potassium chloride to the samples, also due to some extent to the slight absorbance by interfering proteins in the extracts. Figure 2 shows a linear relationship between peak height and the amount of myoglobin in the standards in Fig. 1, and in routine use one standard was sufficient for each chromatogram. Reproducibility was satisfactory; the standard deviation of the mean of duplicates over 14 samples of bovine muscle was ? 0.125 mg/g for an overall mean myoglobin concentration of 4.08 mg/g. PAUL ARC Meat Research Institute, Bristol BSl8 7 DY. England Received January 31. 1978
D. WARRISS
Lay&d
REFERENCES Warriss, P. (1976) Anal. Biochem. 72, 104. Coulson, A. F. W. (1972) Anal. B&hem. 49, 589. 3. Akeson, A.. Biorck, G.. and Simon, R. (1968) Acta Med. I. 2.
Stand.
183, 307.
