Myoglobin extraction

Apply dialysate to column and resolve myoglobin extract with chromatography elution buffer at a flow rate of 60 ml/hr. 

Native-PAGE (UNIT B3.1) can be used to assess the purity of the myoglobin extract, which should produce a single protein band with a molecular weight of 17.8 kDa. 

A separation of myoglobin and lysozyme has been presented by Nicoud [44]. This purification was performed on SMB containing 8 columns using ACA 54 (Biosepra, France) as support. Very pure extracts (>98%) and raffinates (>98%) were obtained from a 50-50 mixture. An internal profile is given in Fig. 7. Another recently presented example is the separation of cyclosporine A from cyclic oligopeptide and other impurities in the reversed phase mode or by adsorption on silica gel presented by Schulte et al. [10]. 

Apart from differences between muscle tissues from various parts of an animal, there are qualitative and quantitative differences in composition between animal species. Therefore, analytical methods will always have lo be tested on material from each individual species, since differences in fat composition, in the presence of species-specific proteins, and in colored components such as in the case of myoglobin in poultry and beef may influence both the extraction and the separation of the analytes. As an example, a recovery higher than 70% was obtained for furazolidone after spiking chicken and veal calf muscle tissue but only 10% after spiking pork tissue (16). In this study, the recovery from pork meat could markedly be improved by addition to the aqueous exfraction solvent of about 25% acetonitrile, an observation indicating binding of furazolidone to pork-specific proteins. 

The NO-heme extract from uncooked cured meat or model solutions with pure hemoglobin or myoglobin faded rapidly. 

Fading of acetone extracts of pure NO-myoglobin solutions could be prevented by adding 1 ml of fresh 0.5% neutralized cysteine hydrochloride to 9 ml of NO-myoglobin solution. 

Meat discoloration studies typically involve a maximum of 5 days, with discoloration analy-sis being performed every day or on alternate days. The actual experimental time involved in the objective assessment of discoloration is not extensive and depends on the number of samples being analyzed. Colorimetric measurements with hand-held colorimeters are very rapid (three measurements per meat surface in < 1 min). Spectral scans of meat surfaces require 1 to 2 min. Extraction and analysis of ground meat products has the added step of homogenization and filtration prior to spectrophotometry, but relative to many laboratory procedures, this is relatively quick. Isolation and purification of preparative amounts of myoglobin requires only 2 to 3 days once appropriate preparations are made. Finally, metmyoglobin can be reduced to oxymyoglobin in 15 to 20 min. 

Describes a method to calculate the relative concentration of myoglobin, oxymyoglobin, and metmyoglobin present in a meat extract. 

Three analytical procedures are usually applied to the purification of myoglobin from crude meat extracts. 

Fig. 27 HPLC separation of myoglobin and hemoglobin on Bio-Gel TSK Phenyl-5-PW. (A) Standard solution. (B) Ground beef extract. (From Ref. 286.)...

Figure 11.15 Cation-exchange micro-LC analysis of a mixture of model proteins (a) the original sample consisting of myoglobin (M), cytochrome C (C) and lysozyme (L) (b) and (c) proteins adsorbed on to and then released from the polyacrylic acid coated fibre with extraction times of 5 and 240 s, respectively. Reprinted from Journal of Microcolumn Separations, 8, J.-L. Liao et al., Solid phase micro extraction of biopolymers, exemplified with adsorption of basic proteins onto a fiber coated with polyacryfic acid, pp. 1-4, 1996, with permission from John Wiley Sons, New York.

Love and Pearson (6) have described a model meat system in which bovine muscle was ground and extracted with distilled, deionized water at 4°C until it was devoid of color, indicating the removal of all meat pigments, i.e., myoglobin and hemoglobin. Other water-soluble components would also be partially or completely extracted by this procedure. The remaining extracted muscle was then used as a model system to which purified myoglobin, ferrous iron and ferric iron were added back to ascertain their role in WOF. 

O Regan S, Fong JS, Drummond KN (1979) Renal injury after muscle extract infusion in rats absence of toxicity with myoglobin. Experientia 35 805-806... 

---