The Determination 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.
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 4ยฐC. 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 15ยฐC 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,