Rapid separation and quantification of myofibrillar proteins by high performance gel permeation chromatography

Abstract

A gel permeation chromatography method was developed to rapidly separate and quantify myofibrillar proteins from bovine and porcine muscle samples. Muscle samples were homogenized with one of three salt buffers (phosphate buffered saline, 0·015 m NaCl, 0·02 m Na 2HPO 4, pH 7·4; potassium phosphate, 0·03 m KH 2PO 4, pH 7·4; or phosphate buffered potassium iodide, 1·1 m KI, 0·01 m KH 2PO 4, pH 7·4), filtered and separated by the chromatography method developed. Samples were eluted from the column with 0·5 m KCl, 20 m m Tris buffer, pH 6·0 at 0·6 ml/min and detected at 280 nm. A set of standards was dissolved in elution buffer and was used to create a standard curve for molecular weight determinations. Actomyosin, myosin, tropomyosin, actin, myosin light chains and troponin peaks were identified by spiking the samples with standards and by comparison to Phastgel sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Quantitation was achieved either by using regression equations which described the relationship between peak area and concentration or by expression of the protein in the fraction collection representing the peak as a percent of the total protein injected. The method described here was capable of separating the major proteins from a crude muscle homogenate in thirty minutes with very good reproducibility.