In order to study a particular protein in the laboratory, one must usually separate it from all other cell components, including other similar proteins. The first step in protein purification is to prepare a solution of proteins. The source of a protein is often whole cells in which the target protein accounts for less than 0.1% of the total dry weight. Isolation of an intracellular protein requires that cells be suspended in a buffer solution and homogenized, or disrupted into cell fragments. Under these conditions, most proteins dissolve.

The next step in protein purification is often a relatively crude separation, or fractionation, procedure that makes use of the different solubilities of proteins in salt solutions. Ammonium sulfate is frequently used in such fractionations. Enough ammonium sulfate is mixed with the solution of proteins to precipitate the less soluble impurities, which are removed by centrifugation. The target protein and other, more soluble proteins remain in the fluid, called the supernatant fraction. Next, more ammonium sulfate is added to the supernatant fraction until the desired protein is precipitated.

The mixture is centrifuged, the fluid removed, and the precipitate dissolved in a minimal volume of buffer solution. Typically, fractionation using ammonium sulfate gives a two- to threefold purification (i.e., one-half to two-thirds of the unwanted proteins have been removed from the resulting enriched protein fraction). At this point, the solvent containing residual ammonium sulfate is exchanged by dialysis for a buffer solution suitable for chromatography.

In dialysis, a protein solution is placed in a cylinder of cellophane tubing sealed at one end. The tubing is then sealed at the other end and suspended in a large volume of buffer. The cellophane membrane is semipermeable: high-molecular weight proteins are too large to pass through the pores of the membrane so proteins remain inside the tubing while low- molecular-weight solutes (including, in this case, ammonium and sulfate ions) diffuse out and are replaced by solutes in the buffer.

Column chromatography can then be used to fractionate the mixture of proteins that remains after ammonium sulfate precipitation and dialysis. A cylindrical column is filled with an insoluble material such as substituted cellulose fibers or synthetic beads. The protein mixture is applied to the column and washed through the matrix of insoluble material by the addition of solvent. As solvent flows through the column, the eluate (the liquid emerging from the bottom of the column) is collected in many fractions, a few of which are represented in Figure 3.11a.

High-performance liquid chromatography (HPLC) Ion-exchange chromatography Gel-filtration chromatography Affinity chromatography

Electrophoresis separates proteins based on their migration in an electric field. In polyacrylamide gel electrophoresis (PAGE), protein samples are placed on a highly cross-linked gel matrix of polyacrylamide and an electric field is applied.

Mass spectrometry Mass spectrometry, as the name implies, is a technique that determines the mass of a molecule. The most basic type of mass spectrometer measures the time that it takes for a charged gas phase molecule to travel from the point of injection to a sensitive detector. This time depends on the charge of a molecule and its mass and the result is reported as the mass/charge ratio. The technique has been used in chemistry for almost one hundred years but its application to proteins was limited because, until recently, it was not possible to disperse charged protein molecules into a gaseous stream of particles.

This problem was solved in the late 1980s with the development of two new types of mass spectromety. In electrospray mass spectrometry the protein solution is pumped through a metal needle at high voltage to create tiny droplets. The liquid rapidly evaporates in a vacuum and the charged proteins are focused on a detector by a magnetic field. The second new technique is called matrix-assisted desorption ionization (MALDI).

In this method the protein is mixed with a chemical matrix and the mixture is precipitated on a metal substrate. The matrix is a small organic molecule that absorbs light at a particular wavelength. A laser pulse at the absorption wavelength imparts energy to the protein molecules via the matrix. The proteins are instantly released from the substrate (desorbed) and directed to the detector (Figure 3.14). When time-of-flight (TOF) is measured, the technique is called MALDI–TOF.

Once a protein has been isolated, its amino acid composition can be determined. First, the peptide bonds of the protein are cleaved by acid hydrolysis, typically using 6 M HCl (Figure 3.15).

Next, the hydrolyzed mixture, or hydrolysate, is subjected to a chromatographic procedure in which each of the amino acids is separated and quantitated, a process called amino acid analysis. One method of amino acid analysis involves treatment of the protein hydrolysate with phenylisothiocyanate (PITC) at pH 9.0 to generate phenylthiocarbamoyl (PTC)–amino acid derivatives (Figure 3.16).

The PTC–amino acid mixture is then subjected to HPLC in a column of fine silica beads to which short hydrocarbon chains have been attached. The amino acids are separated by the hydrophobic properties of their side chains. As each PTC–amino acid derivative is eluted, it is detected and its concentration is determined by measuring the absorbance of the eluate at 254 nm (the peak absorbance of the PTC moiety). A plot of the absorbance of the eluate as a function of time is given in Figure 3.17.

Table 3.3 shows the average frequency of amino acid residues in more than 1000 different proteins whose sequences are deposited in protein databases.

Determining the Sequence of Amino Acid Residues Amino acid analysis provides information on the composition of a protein but not its primary structure (sequence of residues). In 1950 Pehr Edman developed a technique that permits removal and identification of one residue at a time from the N-terminus of a protein. The Edman degradation procedure involves treating a protein at pH 9.0 with PITC, also known as the Edman reagent.

When a protein contains one or more cystine residues, the disulfide bonds must be cleaved to permit release of the cysteine residues as PTH– amino acids during the appropriate cycles of Edman degradation. Thiol compounds, such as 2-mercaptoethanol, are often used to cleave disulfide bonds. Thiols reduce cystine residues to pairs of cysteine residues (Figure 3.19a).

The reactive sulfhydryl groups of the cysteine residues are then blocked by treatment with an alkylating agent, such as iodoacetate, which converts oxidizable cysteine residues to stable S- carboxymethylcysteine residues, thereby preventing the re-formation of disulfide bonds in the presence of oxygen (Figure 3.19b).

Most proteins contain too many residues to be completely sequenced by Edman degradation proceeding only from the N-terminus. Therefore, proteases (enzymesthat catalyze the hydrolysis of peptide bonds in proteins) or certain chemical reagents are used to selectively cleave some of the peptide bonds of a protein. The smaller peptides formed are then isolated and subjected to sequencing by the Edman degradation procedure.

The chemical reagent cyanogen bromide (BrCN) reacts specifically with methionine residues to produce peptides with C-terminal homoserine lactone residues and new N-terminal residues (Figure 3.20).

Many different proteases can be used to generate fragments for protein sequencing. For example, trypsin specifically catalyzes the hydrolysis of peptide bonds on the carbonyl side of lysine and arginine residues, both of which bear positively charged side chains (Figure 3.21a).

Deducing the amino acid sequence of a particular protein from the sequence of its gene (Figure 3.22) overcomes some of the technical limitations of direct analytical techniques.

Comparisons of the Primary Structures of Proteins Reveal Evolutionary Relationships Figure 3.23 Cytochrome c sequences. The sequences of cytochrome c proteins from various species are aligned to show their similarities. In some cases, gaps (signified by hyphens) have been introduced to improve the alignment. The gaps represent deletions and insertions in the genes that encode these proteins. For some species, additional residues at the ends of the sequence have been omitted. Hydrophobic residues are blue and polar residues are red.