Protein acid-base properties

Individual ammo acids differ m their acid-base properties This is important m peptides and proteins where the properties of the substance depend on its ammo acid constituents especially on the nature of the side chains It is also important m analyses m which a complex mixture of ammo acids is separated into its components by taking advantage of the differences m their proton donating and accepting power... 

Electrophoresis is used primarily to analyze mix tures of peptides and proteins rather than individual ammo acids but analogous principles apply Because they incorporate different numbers of ammo acids and because their side chains are different two pep tides will have slightly different acid-base properties and slightly different net charges at a particular pH Thus their mobilities m an electric field will be differ ent and electrophoresis can be used to separate them The medium used to separate peptides and proteins is typically a polyacrylamide gel leading to the term gel electrophoresis for this technique... 

Proteins are the indispensable agents of biological function, and amino acids are the building blocks of proteins. The stunning diversity of the thousands of proteins found in nature arises from the intrinsic properties of only 20 commonly occurring amino acids. These features include (1) the capacity to polymerize, (2) novel acid-base properties, (3) varied structure and chemical functionality in the amino acid side chains, and (4) chirality. This chapter describes each of these properties, laying a foundation for discussions of protein structure (Chapters 5 and 6), enzyme function (Chapters 14-16), and many other subjects in later chapters. 

Noszal, B. Osztas, E., Acid-base properties for each protonation site of six corticotropin fragments, Int. J. Peptide Protein Res. 33, 162-166 (1988). 

ACiD-BASE PROPERTIES OF AMINO ACIDS AND PROTEINS... 

In many problems of peptide sequencing and peptide synthesis it is necessary to be able to separate mixtures of peptides and proteins. The principal methods used for this purpose depend on acid-base properties or on molecular sizes and shapes. 

The acid-base properties, and hence ionic character, of peptides and proteins also can be used to achieve separations. Ion-exchange chromatography, similar to that described for amino acids (Section 25-4C), is an important separation method. Another method based on acid-base character and molecular size depends on differential rates of migration of the ionized forms of a protein in an electric field (electrophoresis). Proteins, like amino acids, have isoelectric points, which are the pH values at which the molecules have no net charge. At all other pH values there will be some degree of net ionic charge. Because different proteins have different ionic properties, they frequently can be separated by electrophoresis in buffered solutions. Another method, which is used for the separation and purification of enzymes, is affinity chromatography, which was described briefly in Section 9-2B. 

Table 5.11. The lonisable Groups which Contribute to the Acid-Base Properties of Proteins, Shown with their Approximate pKa Values...

The amino-acids that make up the primary structure of proteins will change their charge when the pH of the solution is altered due to their acid-base properties (Section 5.3 and Appendix 5.1). The effects of pH on enzyme-catalysed reactions can be complex since both Km and may be affected. Here, only the effects on Kmax are considered, as this usually reflects a single constant rather than several that may be associated within the constant Km (see Section 5.4.4.). It is assumed that pH does not change the limiting step in a multi-step process and that the substrate is saturating at all times. 

Stereochemistry of Amino Acids 339 Acid-Base Properties of Amino Acids 339 The Isoelectric Point 342 Electrophoresis 343 The Peptide Bond 343 Primary Structure of Proteins 344 Secondary Structure of Proteins 344 Tertiary Structure of Proteins 345 The Folding Problem 346 Denaturing Proteins 346 Enzymes 347... 

Separation of proteins based on differences in their electrical charge depends on their acid-base properties, which are mostly determined by the number and type of ionizable side groups in the peptide chain. Since proteins are different from each other with respect to their composition and amino acid sequence, they also have distinct acid-base properties. Information on these properties allows a prediction of the behavior of a given protein when exposed to an electrical field. 

The acid-base properties of proteins are exploited in two methods, electrophoresis and ion exchange chromatography. These are widely used in the analysis and separation of protein mixtures. 

A mammalian cell may contain as few as 2000 different proteins and as many as 50,000 at any given time. Each of these is uniquely suited to the function it performs, and this in turn depends on its size, shape, solubility in aqueous media, acid-base properties, propensity to form fibers, and numerous other physical and chemical properties. The component amino acids are largely responsible for the ability of proteins to perform their biologic roles. The properties of amino acids are therefore of paramount importance in determining how proteins work. 

The only method not based on acid-base properties of proteins is gel filtration chromatography, which takes advantage of protein size. 

An alternative and possibly more effective way is the determination of the change in the acid-base property of the protein itself. If the binding of a substance to a protein can significantly change this behavior, the detection of this change enables the construction of a new type of biosensor. This chapter focuses on the use of ISFETs for the determination of the protein acid-base behavior. 

The acid-base behavior of proteins can reveal some important properties with respect to both their composition (selectivity) and their concentration (sensitivity). The most direct way to exploit these acid-base properties is to make use of acid-base titration, Titrant should be added somehow and the resulting change in pH should be measured. Since the ion-sensitive field-effect transistor (ISFET) is suitable for fast (and local) pH detection, an ISFET can be used for protein titration if the protein to be detected can be immobilized in a membrane, deposited on top of the device. Advantages are the small amount of protein necessary for the characterization owing to the small membrane volume, and the relatively short time needed to perform a full titration. 

In this section, four quite different methods of protein determination are described. Some of these methods mainly give qualitative information, e.g a protein-specific signature of the solution, while others directly yield quantitative data about the protein to be detected. All methods have in common that they somehow rely on the acid-base properties of proteins and use (modified) ISFETs as transducing elements. 

As shown in Table IVA, the cells bind approximately 5% of the total radioactivity added 32% of this eictivity is associated with the cellular lipid fraction and 64% with the nonlipid (presumably protein) fraction. Of the lipid-associated activity, the distribution of I into cellular phospholipids, lysophosphatides, triacylglycerides, and free fatty acids is shown in Table IVB. The iodination reaction labels a variety of lipids of all classes without preference to charge, acid-base properties, or number of fatty acyl constituents, appears not to label intracellular lipids (e.g., cardiolipin, a major mitochondrial component), and labels lipids on both the exterior and interior hydrophilic surfaces of the plasma membrane (i.e., phosphatidylcholine and sphingomyelin as well as phosphatidyleth-anolamine and phosphatidylserine) (Table IVB). ... 

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