O Proteins And Proteinlike Compounds

The chemistry of proteins is complex, with many facets not completely understood. Protein structure is usually studied in basic organic chemistry and, to a greater extent, in biochemistry, but for the purposes of this chapter, some of the more important topics are summarized, with emphasis on relationships to medicinal chemistry. Much progress has been made in understanding the more sophisticated features of protein structure4 and its correlation with physicochemical and biological properties. With the total synthesis of ribonu-clease in 1969, new approaches to the study of SARs among proteins have involved the synthesis of modified proteins.

Many types of compounds important in medicinal chemistry are classified structurally as proteins, including enzymes, antigens, and antibodies. Numerous hormones are low-relative-molecular-mass proteins and so are called simple proteins. Fundamentally, all proteins are composed of one or more polypeptide chains; that is, the primary organizational level of protein structure is the polypeptide (polyamide) chain composed of naturally occurring amino acids bonded to one another by amide linkages (Fig. 27.1). The specific physicochemical and biological properties of proteins depend not only on the nature of the specific amino acids and their sequence within the polypeptide chain but also on conformational characteristics.

Conformational Features of Protein Structure

As stated, the polypeptide chain is considered to be the primary level of protein structure, and the folding of the polypeptide chains into a specific coiled structure is maintained through hydrogen-bonding interactions (intramolecular) (Fig. 27.2). The folding pattern is the secondary level of protein structure. The intramolecular hydrogen bonds involve the partially negative oxygens of amide carbonyl groups and the partially positive hydrogens of the amide —NH. Additional factors, such as ionic bonding between positively and negatively charged groups and disulfide bonds, help stabilize such folded structures.

The arrangement and interfolding of the coiled chains into layers determine the tertiary and higher levels of protein structure. Such final conformational character is determined by various types of interaction, primarily hydrophobic forces and, to some extent, hydrogen bonding and ion pairing.4,5 Hydrophobic forces are implicated in many biological phenomena associated with protein structure and interactions.6 The side chains (R groups) of

Figure 27.1 • Diagrammatic representation of a fully extended polypeptide chain with the bond lengths and the bond angles derived from crystal structures and other experimental evidence. (From Corey, R. B., and Pauling, L.: Proc. R. Soc. Lond. Ser. B 141:10, 1953.)

various amino acids have hydrocarbon moieties that are hydrophobic, and they have minimal tendency to associate with water molecules, whereas water molecules are strongly associated through hydrogen bonding. Such hydrophobic R groups tend to get close to one another, with exclusion of water molecules, to form "bonds" between different segments of the chain or between different chains. These are often termed hydrophobic bonds, hydrophobic forces, or hydrophobic interactions.

The study of protein structure has required several physico-chemical methods of analysis.4 Ultraviolet spectrophotometry has been applied to the assessment of conformational changes that proteins undergo. Conformational changes can be investigated by the direct plotting of the difference in absorption of the protein under various sets of conditions.

Figure 27.2 • Left-handed and right-handed a-helices. The R and H groups on the a-carbon atom are in the correct position corresponding to the known configuration of the L-amino acids in proteins. (From Pauling, L., and Corey, R. B.: unpublished drawings.)

X-ray analysis has been most useful in the elucidation of the structures of several proteins (e.g., myoglobulin and lysozyme). Absolute determinations of conformation and helical content can be made by x-ray diffraction analysis. Optical rotation of proteins has also been studied fruitfully. The specific rotations of proteins are always negative, and extreme changes in pH (when the protein is in solution) and conditions that promote denaturation (urea solutions, increased temperatures) tend to augment the negative optical rotation. Accordingly, it is thought that the changes in rotation are caused by conformational changes (i.e., changes in protein structure at the secondary and higher levels of organization). Optical rotatory dispersion has also been used to study conformational alterations and differences among globular proteins. Additionally, circular dichroism methodology has been involved in structural studies. The shape and the magnitude of rotatory dispersion curves and circular dichroism spectra are very sensitive to conformational alterations; thus, the effects of enzyme inhibitors on conformation can be analyzed. Structural studies have included the investigation of the tertiary structures of proteins in high-frequency nuclear magnetic resonance (NMR).7,8

NMR spectroscopy has been of some use in the study of interactions between drug molecules and proteins such as enzymes, proteolipids, and others. NMR has been applied to the study of binding of atropine analogs to acetylcholinesterase9 and interactions involving cholinergic ligands and housefly brain and torpedo electroplax.10 NMR was also used in the determination of the tertiary structure of the cap-sid protein of the human immunodeficiency virus (HIV).11

Factors Affecting Protein Structure

Conditions that promote the hydrolysis of amide linkages affect protein structure (see under "Protein Hydrolysates" previously in this chapter). The highly ordered conformation of a protein can be disorganized (without hydrolysis of the amide linkages), and in the process, the protein's biological activity is lost. This process, customarily called denaturation, involves unfolding of the polypeptide chains, loss of the native conformation of the protein, and disorganization of the uniquely ordered structure, without the cleavage of covalent bonds (e.g., cooked egg albumin). The rupture of native disulfide bonds is usually considered a more extensive and drastic change than denaturation. Criteria for the detection of denaturation involve detection of previously masked —SH, imidazole, and —NH2 groups; decreased solubility; increased susceptibility to the action of proteolytic enzymes; decreased diffusion constant and increased viscosity of protein solution; loss of enzymatic activity if the protein is an enzyme; and modification of antigenic properties.

Purification and Classification

It might be said that it is old-fashioned to classify proteins according to the following system, since so much progress has been made in understanding protein structure. Nevertheless, an outline of this system of classification is given because the terms used are still found in the pharmaceutical and medical literature. Table 27.1 includes the classification and characterization of simple proteins. Before classification, the protein material must be purified as much as possible, which is a very challenging task. Several criteria are used to determine homomolecularity, including crys-tallinity, constant solubility at a given temperature, osmotic pressure in different solvents, diffusion rate, electrophoretic mobility, dielectric constant, chemical assay, spectropho-tometry, and quantification of antigenicity. The methodology of purification is complex; procedures can involve various techniques of chromatography (column), electrophoresis, ul-tracentrifugation, and others. High-performance liquid chro-matography (HPLC) has been applied to the separation of peptides (e.g., the purification of some hypothalamic pep-tides by a combination of chromatographic methods including HPLC).1213

Conjugated proteins contain a nonprotein structural component in addition to the protein moiety, whereas simple proteins contain only the polypeptide chain of amino acid units. Nucleoproteins are conjugated proteins containing nucleic acids as structural components. Glycoproteins are carbohydrate-containing conjugated proteins (e.g., thyroglobu-lin). Phosphoproteins contain phosphate moieties (e.g., casein). Lipoproteins are lipid bearing. Metalloproteins have some bound metal. Chromoproteins, such as hemoglobin or cytochrome, have some chromophoric moiety.

Properties of Proteins

The classification in Table 27.1 is based on solubility properties. Fibrous proteins are water insoluble and highly resistant to hydrolysis by proteolytic enzymes; the collagens, elastins, and keratins are in this class. Globular proteins (albumins, globulins, histones, and protamines) are relatively water soluble; they are also soluble in aqueous solutions containing salts, acids, bases, or ethanol. Enzymes, oxygen-carrying proteins, and protein hormones are globular proteins.

Another important characteristic of proteins is their am-photeric behavior. In solution, proteins migrate in an electric field, and the direction and rate of migration are a function of the net electrical charge of the protein molecule, which in turn depends on the pH of the solution. The isoelectric point is the pH value at which a given protein does not migrate in an electric field; it is a constant for any given protein and can be used as an index of characterization. Proteins differ in rate of migration and in their isoelectric points. Electrophoretic analysis is used to determine purity and for quantitative estimation because proteins differ in elec-trophoretic mobility at any given pH.4

Because they are ionic in solution, proteins bind with cations and anions depending on the pH of the environment. Sometimes, complex salts are formed, and precipitation takes place (e.g., trichloroacetic acid is a precipitating agent for proteins and is used for deproteinizing solutions).

Proteins possess chemical properties characteristic of their component functional groups, but in the native state, some of these groups are "buried" within the tertiary protein structure and may not react readily. Certain denaturation procedures can expose these functions and allow them to respond to the usual chemical reagents (e.g., an exposed —NH2 group can be acetylated by ketene; —CO2H can be esterified with diazomethane).

Color Tests and Miscellaneous Separation and Identification Methods

Proteins respond to the following color tests: (a) biuret, pink to purple with an excess of alkali and a small amount of copper sulfate; (b) ninhydrin, a blue color when boiled with nin-hydrin (triketohydrindene hydrate), which is intensified by the presence of pyridine; (c) Millon test for tyrosine, a brick-red color or precipitate when boiled with mercuric nitrate in an excess of nitric acid; (d) Hopkins-Cole test for trypto-phan, a violet zone with a salt of glyoxylic acid and stratified over sulfuric acid; and (e) xanthoproteic test, a brilliant

TABLE 27.1 Simple (True) Proteins

Class

Characteristics

Occurrence

Albumins

Soluble in water, coagulable by heat and reagents

Egg albumin, lactalbumin, serum albumin, leucosin of

Was this article helpful?

0 0
Free Yourself from Panic Attacks

Free Yourself from Panic Attacks

With all the stresses and strains of modern living, panic attacks are become a common problem for many people. Panic attacks occur when the pressure we are living under starts to creep up and overwhelm us. Often it's a result of running on the treadmill of life and forgetting to watch the signs and symptoms of the effects of excessive stress on our bodies. Thankfully panic attacks are very treatable. Often it is just a matter of learning to recognize the symptoms and learn simple but effective techniques that help you release yourself from the crippling effects a panic attack can bring.

Get My Free Ebook


Post a comment