Amino Acids

Variations among amino acid sequences are responsible for defining proteins. Small variations create a family of proteins such as the myosins; greater variations create different proteins altogether. Because distinctions among proteins can be so slight, it can be difficult to determine if two proteins from different tissues are the same. For example, it remains as yet unproven that lactalbumin (milk) and serum albumin (blood) are the same protein(s) in a given animal. What are the problems? Experimentally, we may have to work with two proteins in equivalent solutions although their natural solutions are different; there may have been different inhibitors, different ionic strengths, and possibly different counterions in the natural solutions. All of these factors may change the initial folding of proteins and their subsequent behavior when altered as we try to isolate them.

There are other problems in distinguishing two proteins, even when we can show a high degree of similarity in physical behavior. Recent evidence indicates that different versions of the same protein are synthesized at different times for different purposes, that is, they come from different genes and may have one or two amino acid changes. For example, the muscle protein myosin has both a fetal and an adult type for each fiber type (e.g., slow and fast muscle) within a species. There are also different myosins in smooth muscle and cardiac muscle. Thus, even if we prove that two proteins are essentially the same, we still must obtain the complete sequence or at least its “fingerprints” (see Chapter 8) to prove that the proteins are identical. We must then prove directly whether one protein migrated (e.g., from the blood into the milk) or whether each protein was produced independently.

Table 3-I offers some of the numerical properties of amino acids. The first column lists the amino acids, emphasizing those that are coded by the genetic code. Only two modified amino acids are included: cystine, the cross-linking of two cysteines (often between two different polypeptide chains); and hydroxyproline, a modified form of proline that is extremely prevalent in collagen, the single most common protein in animals.

The second column gives the official molecular formula, which is shown here as a neutrally charged compound neutralized by hydrogen (H) ions. There are, for example, no sodium (Na+) or chlorine (Cl–) ions neutralizing any charge, although this might occur in a real solution. Thus, this table shows amino acids in an idealized chemical form. When we calculate molecular weights carefully, we will need to account for the hydrogen ions that come on and off at different pH values. If we do not ignore ionized free hydrogen (H+), the molecular weight of the amino acid will be 1 or 2 units more or less than the number given. Depending on how the problem is phrased and how we are attacking it, we may also have to consider the contribution to the molecular weight of those counterions other than H+ that may be present to ensure overall charge neutrality (Fig. 3-1).

In Table 3-I, we distinguish amino acids from the amino acid residues in proteins. In the formation of the peptide bond, we must take into account the loss of one water molecule per bond. For example, if we have a decamer (10 amino acids), 9 water molecules are lost in forming the peptide from the amino acids. Thus, the sum of the molecular weights of the amino acids in a protein is greater than the molecular weight of the protein.

We also note, in Table 3-I, a large range in the molecular mass of amino acids, from glycine at 75 to tryptophan at 204 daltons. (The dalton is equivalent to the mass of one hydrogen atom.) As this is a fairly wide range, there are times when, for certain mathematical considerations, we must take a weighted average based on the relative amounts generally present in proteins. For example, a generally accepted value of 115 daltons is used in optical rotatory dispersion (ORD) calculations, which is obviously an arbitrary number.

Note that there will often be instances in which arbitrary numbers must be used although they cause ambiguity in the results. Generally, the accuracy (but not the precision) of the results suffers, but not necessarily by much. Although the resultant inaccuracy need not cause concern in most cases, we must still be careful not to misinterpret results when using such arbitrary numbers.

The fourth column in Table 3-I shows the percentage of nitrogen in each amino acid. Once again, we note the wide range of nitrogen concentration, from a minimum of 7.7% in tyrosine to a maximum of 32% in arginine; this can have a significant effect on our calculations. For example, if we are measuring the amount of polyarginine (using the Kjeldahl method), which has an average of 35.9% nitrogen (polymeric arginine minus the water), the Kjeldahl conversion factor is 2.8 instead of 6.25. Our protein calculations for polyarginine could therefore be off by a factor of more than 2!

Table 3-II includes other properties of amino acids. Amino acids can be classified by their side chain chemistry, for example, as aliphatic hydrocarbons, alcohols, or acids. The aliphatic hydrocarbons do not have “reactive” side chains, although these side chains do participate in hydrophobic bond formation and therefore cannot be considered inert. All the other amino acids have reactive side chains, each with its own chemistry. Each amino acid has an isoelectric point that is a reflection of the individual pK values of the reactive group. (We will come back to this in Chapter 4.)

All amino acids except glycine have at least one asymmetric carbon atom. There are a few amino acids that have an ultraviolet (UV) absorption, the aromatic amino acids phenylalanine, tyrosine, and tryptophan. Figure 3-2 illustrates the absorption spectra of the three aromatic amino acids in the UV region. We characterize the absorptions of proteins and amino acids by the wavelength of maximum absorption and an intensity factor (that we will discuss in Chapter 10). Thus, if a protein has none of these amino acids, it may not have a spectrum in this part of the UV; but if the protein does have these amino acids, the spectrum can be an extremely helpful and simple technique for monitoring proteins either quantitatively or qualitatively. We should note, too, that the maximum wavelength of the absorption can move over a fairly narrow range with changes in the environment; these changes are usually studied by difference spectroscopy (see Chapter 23).

Let us examine the structure of an amino acid more carefully. We see that most amino acids except proline and hydroxyproline can be written as follows:

The central carbon atom is called the α carbon. The R group represents the side chain. Following general practice, this compound is represented as a charged dipolar compound, or a “zwitterion,” which is the form of the amino acid that exists at pH 7. The evidence for this zwitterion will be...