Human Biochemistry, Volume 2 - Murray R. 1993

Special Topics
Blood Plasma and Coagulation
Plasma Proteins

The total plasma protein concentration is 7–7.5 g/dL (g%), meaning that Proteins constitute the major portion of plasma solids. Plasma proteins form a highly complex mixture comprising not only simple proteins but also mixed or conjugated molecules, such as Glycoproteins and various types of Lipoproteins.

Fractionation of this complex protein mixture into individual components is achieved using Solvents and/or electrolytes, separating various protein fractions based on their solubility. This property forms The basis of so-called salting-out techniques frequently used in clinical laboratories. Plasma proteins are precipitated at varying concentrations of sodium sulfate or ammonium sulfate, allowing them to be divided into three primary groups: fibrinogen, albumin, and globulin.

Blood Plasma is, by definition, an intravascular fluid. In the arterial microcirculation, the intravascular hydrostatic pressure generated by The Heart and large vessels exceeds the interstitial hydrostatic pressure in Tissues by 20–25 mmHg. The leakage of excessive fluid from vessels into the extravascular tissue space is counteracted by the intravascular colloid osmotic pressure exerted by plasma proteins.

Albumin

Albumin is present in plasma at a higher concentration than the other two major proteins, yet it has the lowest molecular weight (Fig. 55.1). This protein provides the primary contribution to intravascular colloid osmotic pressure. Synthesized in the Liver, albumin consists of a single polypeptide chain of 610 Amino Acids. Alongside its role in maintaining colloid osmotic pressure, albumin Functions as a versatile carrier molecule, transporting bilirubin, Fatty acids, numerous drugs, and various Trace Elements present in plasma. Some of its Ligand-binding sites are highly specific and saturable, whereas others exhibit these properties to a much lesser degree. Hypoalbuminemia (low serum albumin concentration), which accompanies liver and Kidney diseases, leads to soft tissue edema due to decreased intravascular colloid osmotic pressure.

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Fig. 55.1. Relative sizes and molecular weights of blood protein molecules (Oncley).

Globulins

As noted in Chapter 5, globulins are proteins insoluble in Water but soluble in salt solutions. Serum globulins comprise a heterogeneous, complex mixture of protein molecules commonly designated as a-, ß-, and y-globulins (sometimes with additional numerical subscripts); this Classification is based on electrophoretic mobility (Fig. 55.2). A more rational classification relies on the Structure AND FUNCTIONS of globulins.

Glycoproteins contain covalently linked oligosaccharide moieties (see Chapter 54). These proteins are found within the a₁- and a₂-globulin fractions. Among glycoproteins are many specialized proteins whose functions have been characterized to varying degrees.

Lipoproteins contain Lipids, typically noncovalently associated with the protein molecule (see Chapter 26). During Electrophoresis, lipoproteins migrate alongside a- or ß-globulins. The higher the lipid content and the lower the protein content of a lipoprotein, the lower its specific gravity. Lipoproteins serve as carriers for various lipids and compounds that dissolve in them rather than in the aqueous plasma phase.

Certain metal-binding proteins, such as transferrin, exhibit globulin-like properties and transport trace elements present in the plasma.

Normal plasma contains A number of Enzymes, notably Phosphatases, lipases, Lactate dehydrogenase, amylase, and ferroxidase (ceruloplasmin). Upon tissue destruction or membrane disruption, intracellular enzymes are released into the intravascular space, where their catalytic activity can serve as a quantitative indicator of the extent of tissue damage. In clinical medicine, the determination of serum transaminases, creatine Kinases, and acid phosphatases is of particular importance.

Polypeptide Hormones circulate within the plasma. Hydrophobic Steroids and 1,25-dihydroxyvitamin D₃ circulate in a bound form (i.e., transported by specific carrier proteins).

Important plasma components include IMMUNOGLOBULINS, which act as effectors of the humoral immune system, and fibrinogen, the precursor of fibrin that forms blood clots. Both classes of plasma proteins will be discussed in greater detail below.

Plasma lipoproteins are discussed in Chapter 26.

Fig. 55.2. Cellulose acetate zone electrophoresis. A. A small amount of serum or other fluid is applied to a cellulose acetate strip. B. Electrophoresis of the sample is performed in a buffer solution. C. Protein bands are visualized by staining. D. Densitometric scanning of the cellulose acetate strip reveals peaks corresponding to albumin, a₁-globulin, a₂-globulin, ß-globulin, and y-globulin on the densitogram. (Reproduced, with permission, from Stites D.P., Stobo J. D., Wells J. V. (eds). Basic and Clinical Immunology, 6th ed. Appleton and Lange, 1987.)

Immunoglobulins

Immunoglobulins, or Antibodies, are synthesized by B lymphocytes or their derivative plasma Cells. Antibodies bind to antigenic determinants of other molecules with remarkable Specificity.

All immunoglobulin molecules consist of two identical light (L) chains (molecular weight 23,000) and two identical heavy (H) chains (molecular weight 53,000–75,000) assembled into a tetramer (L₂H₂) via Disulfide Bonds (Fig. 55.3). Each chain can be conceptually divided into specific domains or regions with distinct Structural and functional significance. The half of the light chain containing the carboxyl terminus is termed the constant region (C_L), whereas the amino-terminal half is the variable region (V_L). Approximately the N-terminal quarter of the heavy chain constitutes the variable region of the H chain (V_H), while the remaining three-quarters comprise the constant regions (C_H1, C_H2, C_H3). The antigen-binding site of an immunoglobulin is formed by the N-terminal variable regions of both light and heavy chains, namely the V_H and V_L domains. Rather than being mere linear Amino acid sequences, these domains form globular structures with secondary and tertiary architecture that ensure efficient binding to specific Antigens. As illustrated in Fig. 55.3, Enzymatic Cleavage of the immunoglobulin molecule with Papain yields two antigen-binding fragments (Fab) and one crystallizable fragment (Fc). The segment located between the C_H1 and C_H2 domains is referred to as the hinge region.

There are two primary types of light chains—kappa (k) and lambda (λ)—which differ in their C_L region structure (Table 55.1). An individual immunoglobulin molecule contains either two k chains or two λ chains, but never both simultaneously. Human immunoglobulins predominantly incorporate k chains.

Five classes of heavy (H) chains have been identified in humans (Table 55.1), distinguished by their C_H regions. These classes are designated by the Greek letters y, a, μ, δ, and ε, with molecular weights ranging from 50,000 to 75,000 (Table 55.1). The μ and ε chains contain four constant domains, whereas other chains contain three. The heavy chain type determines the immunoglobulin class and, consequently, its effector functions. There are five immunoglobulin classes: IgG, IgA, IgM, IgD, and IgE. As shown in Table 55.1, several heavy chain classes can be subdivided into subclasses based on minor structural differences within the C_H regions.

The variable regions of immunoglobulins, composed of V_L and V_H domains, exhibit extensive heterogeneity. In fact, no two variable regions from different individuals share an identical Amino Acid Sequence. Nevertheless, certain regions share structural similarities and can be categorized into three groups based on amino acid Sequence Homology: the V_к group for k chains, the V_λ group for λ chains, and the V_H group for H chains. At higher resolution, subgroups can be distinguished within each of these three main groups. Thus, the variable region framework contains several relatively invariant segments. Comparison of variable regions across different light or heavy chains reveals that interspersed among the relatively invariant regions (which define groups and subgroups) are hypervariable segments (Fig. 55.4). Light chains contain three such segments, whereas heavy chains possess four.

Fig. 55.3. Simplified model of a human IgG antibody molecule, showing the four-chain basic structure and domains. V, variable region; C, constant region; vertical arrow, hinge region. Bold lines indicate H and L chains; thin lines indicate disulfide bonds. (Modified and reproduced, with permission, from Stites D. P., Stobo J. D., Wells J. V. (eds.). Basic and Clinical Immunology, 6th ed. Appleton and Lange, 1987.)

Table 55.1. Properties of human immunoglobulin chains. (Reproduced, with permission, from Stites D. P., Stobo J. D., Wells J. V. (editors). Basic and Clinical Immunology, 6th ed., Appleton and Lange, 1987.)

¹⁾ Formerly Inv (1)-(3).

²⁾ For y3, 60,000.

The constant regions of immunoglobulin molecules, particularly C_H2 and C_H3 (as well as C_H4 in IgM and IgE), which form the Fc fragment, are responsible for class-specific EFFECTOR FUNCTIONS OF immunoglobulins (Table 55.2). Some immunoglobulins, such as IgG, exist solely in a monomeric form. Other immunoglobulins (IgA and IgM) can form Higher-order oligomers comprising two or three (IgA) or five (IgM) monomers (Fig. 55.5).

Fig. 55.4. Schematic model of an IgG molecule showing the approximate locations of hypervariable regions in the heavy and light chains. (Modified and reproduced, with permission, from Stites D. P., Stobo J. D., Wells J. V. (eds.). Basic and Clinical Immunology. 6th ed. Appleton and Lange. 1987.)

Table 55.2. Properties of human immunoglobulins. (Reproduced, with permission, from Stites D. P., Stobo J. D., Wells J. V. (editors): Basic and Clinical Immunology, 6th ed. Appleton and Lange, 1987.)

⁺ For monomeric serum IgA

+ J chain

’ Secretory component

** For secretory IgA

Fig. 55.5. Schematic representation of human immunoglobulin polymer molecules. Polypeptide chains are indicated by bold lines; interchain disulfide bonds by thin lines. (Reproduced, with permission, from Stites D. P., Stobo J. D., Wells J. V. (eds.). Basic and Clinical Immunology, 6th ed. Appleton and Lange, 1987.)

Heavy and light chains are synthesized as separate molecules and subsequently assembled into mature immunoglobulins within B cells or plasma cells; all immunoglobulins are, without exception, glycoproteins (Table 55.1).

Each immunoglobulin light chain is the product of at least three distinct structural genes: a variable region Gene (V_L), a joining region gene (J) (unrelated to the J chain of IgA or IgM), and a constant region gene (C_L). Each heavy chain is the product of at least four distinct genes: a variable region gene (V_H), a diversity gene (D), a joining region gene (J), and a constant region gene (C_H). Thus, the classical "one gene, one protein" dogma does not apply in this case. The molecular mechanisms responsible for the synthesis of individual immunoglobulin chains from multiple structural genes are discussed in Chapters 38 and 41.

Each individual is capable of synthesizing antibodies against approximately one million different antigens. This antibody diversity is determined by combinations of various structural genes involved in forming each of the immunoglobulin chains, as well as by a high frequency of somatic Mutations in the GENES OF THE V_H and V_L regions.

In most immune responses, antibodies of identical specificity but different classes are produced following the administration of an immunogen (immunizing antigen) in a strictly chronological order. A single type of antigen-specific immunoglobulin light chain can combine with an antigen-specific μ heavy chain to form an IgM molecule. Later, the same antigen-specific light chain combines with a у heavy chain possessing an identical variable V_H region to form an IgG immunoglobulin molecule with the same antigen specificity as the IgM molecules. Subsequently, this same light chain may associate with an a heavy chain containing an identical V_H region, forming an IgA molecule with an antigen specificity analogous to that of the IgG molecule. These three classes of immunoglobulins (IgM, IgG, and IgA), synthesized in response to the same antigen, share identical variable domains in their light (V_L) and heavy (V_H) chains and are referred to as idiotypic (idiotypes). Different classes of isotypes occur when different C_H regions combine with the same antigen-specific V_H region. Chapter 41 discusses the genetic regulatory mechanisms responsible for C_H region gene switching.

Certain diseases are characterized by an increased production of specific classes or even specific molecules of immunoglobulins, as is the case in clonal plasma Cell malignancies (such as multiple myeloma). Conversely, hypogammaglobulinemia involves a reduced synthesis of either a single immunoglobulin class (e.g., IgA or IgG) or multiple classes combined (IgA, IgD, IgE, IgG, IgM). In almost all cases, variations in immunoglobulin levels result from alterations in either The rate of synthesis or the secretion of these molecules. The causes of such alterations are highly diverse.