Isolating a protein may be compared to playing a game of golf. In golf, the player is faced with a series of problems, each unique and yet similar to problems previously encountered. In facing each problem the player must analyse the situation and decide, from experience, which club is likely to give the best result in the given circumstances. Similarly, in attempting to isolate proteins, researchers face a series of similar-yetunique problems. To solve these they must dip into their bags and select an appropriate technique. The purpose of this book is thus to fill the beginnerís “golf bag” with techniques relevant to protein isolation, hopefully to improve their game.
Developing a protein isolation is also somewhat like finding a route up a mountainside. Different routes have to be explored and base-camps established at each stage. Occasionally it will be necessary to return to the base of the mountain for further supplies, and haul these up to the established camps, before the next stage can be attacked. A successful climb is always rewarding and if an efficient route is established, it may become a pass, opening the way to further discoveries.
1.1 Why do it?
This book is about the methods that biochemists use to isolate proteins, and so it may be asked, “why isolate proteins?” Looked at in one way, living organisms may be regarded as machines with features in common with the entities that we commonly think of as “machines”. A typical machine is made of a number of parts which interact, transduce energy, and bring about some desired effect. Mechanical machines have moving parts, while electronic machines move electrons. “Engines” convert energy to mechanical motion. Internal combustion engines, for example, convert chemical energy to mechanical motion. Similarly, living organisms such as the human body are complex machines made up of many interacting systems. Proteins constitute the majority of the working parts of these systems and there are thus diverse reasons for isolating proteins, viz.;
• To gain insight. As with any mechanism, to study the way in whicha living system works it is necessary to dismantle the machine and to isolate the component parts so that they may be studied, separately and in their interaction with other parts. The knowledge that is gained in this way may be put to practical use, for example, in the design of medicines, diagnostics, pesticides, or industrial processes.
For use in Medicine. Many proteins may themselves be used as“medicines” to make up for losses or inadequate synthesis. Examples are hormones, such as insulin, which is used in the therapy of diabetes, and blood fractions, such as the so-called Factor VIII, which is used in the therapy of haemophilia. Other proteins may be used in medical diagnostics, an example being the enzymes glucose oxidase and peroxidase, which are used to measure glucose levels in biological fluids, such as blood and urine.
• For use in Industry. Many enzymes are used in industrial processes,especially where the materials being processed are of biological origin.In every case a pure protein is desirable as impurities may either be misleading, dangerous or unproductive, respectively. Protein isolation is,therefore, a very common, almost central, procedure in biochemistry.
1.2 Properties of proteins that influence the methods used in their study
It must be appreciated that proteins have two properties which determine the overall approach to protein isolation and make this different from the approach used to isolate small natural molecules.
Proteins are labile. As molecules go, proteins are relatively large and delicate and their shape is easily changed, a process called denaturation, which leads to loss of their biological activity. This means that only mild procedures can be used and techniques such as boiling and distillation, which are commonly used in organic chemistry, are thus verboten.
Proteins are similar to one another. All proteins are composed of essentially the same amino acids and differ only in the proportions and sequence of their amino acids, and in the 3-D folding of the amino acid chains. Consequently processes with a high discriminating potential are needed to separate proteins.
1.3 The conceptual basis of protein isolation
In a protein isolation one is endeavouring to purify a particular protein, from some biological (Cell ular) material, or from a bioproduct, since proteins are only synthesised by living systems. The objective is to separate the protein of interest from all non-protein material and all other proteins which occur in the same material. Removing the other proteins is the difficult part because, as noted above, all proteins are similar in their gross properties. In an ideal case, where one was able to remove the contaminating proteins, without any loss of the protein of interest, clearly the total amount of protein would decrease while the activity (which defines the particular protein of interest) would remain the same (Fig. 1 .).
Figure 1. A schematic representation of a protein isolation
Initially (Fig. 1A) there is a small amount of the desired protein “a” and a large amount of total protein “b”. In the course of the isolation, ìbî is reduced and ultimately (Fig. 1B) only ìaî remains, at which point “a”=“b”. Ideally, the amount of “a” remains unchanged but, in practice, this is seldom achieved and less than 100% recovery of purified protein is usually obtained.