Microburger Biochemistry: Extraction and Spectral Characterization of Myoglyobin from Hamburger

Abstract

This experiment provides a demonstration of useful biochemical methods at a Basic or Advanced Level, depending upon the available spectrophotometric equipment. The protocol combines protein extraction, ox-i-dation and reduction, and simple spectroscopic analysis, as well as gel filtration chromatography and generation/analysis of spectral scans. Mammalian myoglobin (Mb) is a monomeric O2-binding protein that functions in muscle to store oxygen. The single iron protoporphyrin IX (heme) group is bound to protein by the amino acid Histidine93. The common, stable forms, Met-Mb and Oxy-Mb are studied because in a non-living system, red Oxy-Mb is converted to brown Met-Mb as bound O2 molecule is released. Mb is easily extracted from steak, to illustrate and address why fresh meat is red and aged meat is brown; the protein has unique spectral properties that are diagnostic for characterization of sample identity. After application of heme redox chemical methods, the MetMb or OxyMb samples can be studied spectroscopically. The color change between Oxy-Mb and Met-Mb is dramatic (illustrating bright red fresh meat vs. brown older meat), and method(s) used in this laboratory are simple, inexpensive, and non-harmful to the student.

Assessment of the purification of a protein by ion exchange and gel permeation chromatography

“…The reduced Mb solution is then diluted 1 to 8 and scanned from 300 to 700 nm. This procedure differs from that of Bylkas and Anderson [9] in that the desalting step is eliminated as the reducing agent does not interfere with the subsequent spectra. As the potassium ferricyanide does interfere with the spectral scans, the oxidized Mb solution is subjected to a desalting step in which the 1.5-ml solution is added to a desalting column (10.0 ml of de-gassed Sigma Sephadex G-25 equilibrated with the 20 mM, pH 5.6, KPi buffer in a 12-cm ϫ 1.5-cm inner diameter Bio-Rad Econo-Pac column).…”

“…Period 1-In the first period the initial isolation of Mb is accomplished by the method of Bylkas and Anderson [9] in which 10.0 g of freshly ground lean hamburger (preferably top round, with its lower fat content) is suspended in 20.0 ml of 20 mM, pH 5.6, potassium phosphate (KPi) 1 buffer. This suspension is centrifuged at 20.0°C for 20 min at 10,000 ϫ g (15,300 rpm with a Beckman JA-14 rotor).…”

“…9). Normally the term specific activity (total units of enzymatic activity/mg of total protein) is used for following the purification of an enzyme.…”

“…of the crude Mb solution (and subsequent purified fractions) is defined as the total mg of Mb divided by the total mg of protein. During the remainder of the period, the students examine the redox properties of Mb with the reducing agent sodium dithionite and the oxidizing reagent potassium ferricyanide that has been extensively described by Bylkas and Anderson [9]. Basically this involves adding to separate 1.5-ml aliquots of the crude Mb supernatant a few small crystals of either sodium dithionite or potassium ferricyanide.…”

“…We feel that for the students to gain a greater appreciation and complete understanding for protein purification, sideby-side comparisons of two methods on the same protein with both activity and protein measurements is warranted. We have accomplished this by extending the work of Bylkas and Andersson [9], which describes the redox properties of myoglobin (Mb). The two purifications methods are evaluated by comparing qualitative evidence (visual inspection of SDS-PAGE gels) to quantitative evidence (Mb and protein determinations).…”