Globulins

Globulins are proteins that are precipitated in both distilled water and 50% saturated ammonium sulfate. This behavior contrasts with that of albumins, which show the opposite characteristics. This operational classification was very important in protein research initially, as these two characteristics were used in purification of various proteins. It is much less important today, but the name globulin is still encountered frequently.

 1. Serum a₁-Globulins

In the electrophoretic fractionation of serum proteins, the a-globulin fraction runs closest to serum albumin. When electrophoresis is carried out in a Veronal buffer of pH 8.6, it is subfractionated into a₁ and a₂ fractions. The former contains a1-antitrypsin (antiproteinase), a1-acid glycoprotein, a ₁-microglobulin, a-fetoprotein, and a-lipoprotein (HDL). The a₂ fraction contains, among others, a ₂-microglobulin (retinol binding protein), a ₂-macroglobulin, a₂-plasmin inhibitor, haptoglobin, and ceruloplasmin.

1.1. a₁-Microglobulin

This glycoprotein (21.76% carbohydrate by weight) is present in plasma at a concentration of 1 mg/100 ml, with a molecular weight of 33,000 and unknown biological functions. It is heterogeneous in electrophoresis, but there is no proven heterogeneity in the amino acid sequence.

1.2. a₁-Antitrypsin, Inter-a-trypsin Inhibitor

 These are two proteinase inhibitors in the a-globulin fraction. They inhibit many proteinases, including trypsin and chymotrypsin, but their roles in homeostasis are not clearly established.

1.3. a-Fetoprotein 

a-Fetoprotein is a protein of unknown function, but structurally related to serum albumin. Its concentration in the serum is high in the fetus but very low in the adult.

1.4. Orosomucoid 

This is also called a1-acid glycoprotein and is a glycoprotein with an unusually high content of carbohydrate, 42% by weight. It may have some evolutionary relationship to immunoglobulins.

 2. Serum a₂-Globulins

2.1. a₂-Microglobulin

 a₂-Microglobulin is also known as retinol binding protein (RBP) and has a molecular weight of, 21,000 consisting of 181 amino acid residues, a sedimentation coefficient of 2.3 S, and no carbohydrates; its structure is known (1). It binds one molecule of retinol (vitamin A) and transports it through the circulation in association with transthyretin (formerly called prealbumin, a homotetramer of 16-kDa subunits) as a 1:1 complex of 85 kDa. Its structure consists of a two-layered up and down b-barrel that encloses a primarily hydrophobic cavity designed to bind retinol. This protein is a member of a large family of ligand transport proteins of similar structures that includes beta-lactoglobulin and a bilin-binding protein.

2.2. a₂-Plasmin Inhibitor

 a₂-Plasmin inhibitor is also known as a₂-antiplasmin and has a molecular weight of 67,000 and carbohydrate content of 12% by weight. It inhibits serine proteinases by forming a covalent bond with the serine residue at their active sites. This protein inhibits the degradation of fibrin clots by plasmin; in individuals who lack this protein, fibrinolysis occurs at an early stage of blood clotting, leading to a premature degradation of fibrin clot and causing serious bleeding. a ₂-plasmin inhibitor obstructs the action of plasmin toward fibrin by binding to specific regions of plasmin and its precursor plasminogen.

2.3. a₂-Macroglobulin

 This is a homo-tetrameric glycoprotein (7% carbohydrates) of 725,000 that inhibits almost any kind of proteinase, including those belonging to the four classes of serine, thiol, metallo, and carboxyl proteinases (2). The mechanism of inhibition is entirely different from that of other proteinase inhibitors in that, instead of binding to the active sites of the target enzymes, a ₂-macroglobulin entraps medium-sized proteinases of between approximately 10,000–100,000 molecular weight within its cavity. The protein has a region in its amino acid sequence that is highly vulnerable to proteolytic attack; when one or more peptide bonds are hydrolyzed in this region, a large conformational change takes place to engulf the proteinase inside the molecular cavity and to react it with a highly reactive thiol ester bond of the macroglobulin. The active sites of the entrapped proteinases are not inhibited, and they remain active toward low-molecular-weight substrates, but are inactive toward large substrates, simply as a result of steric hindrance. The altered form of a₂-macroglobulin is rapidly cleared from the blood serum, due to its internalization after binding to the receptor molecule called LRP (standing for low-density-lipoprotein receptor-related protein) on the membrane surface of peripheral cells such as fibroblasts, macrophages, hepatocytes, and so forth.

2.4. Haptoglobin 

This is a glycoprotein (carbohydrate content of about 19% by weight) in the serum with the capacity to bind two half-molecules of free hemoglobin in the serum, as in the case of hemolytic anemia. It is composed of a1 and a 2 chains of 9.1- and 16,000 molecular weight, plus two identical b chains of 40 kDa. Clearance of the haptoglobin-hemoglobin complex from the blood serum has a half-life of 10 to 20 min.

2.5. Ceruloplasmin 

This is a blue-colored copper-containing glycoprotein, with eight copper atoms per molecule, a carbohydrate content of about 8% by weight, and a high molecular weight of 132,000. It migrates in the a₂ region, its sedimentation coefficient is 7 S, and it is made of four heterogeneous subunits.

2.6. Antithrombins 

Antithrombin III is a glycoprotein (15% carbohydrate by weight) with a molecular weight of 65,000. Its concentration in plasma is 0.2 g/l. It inhibits thrombin, activated forms of blood clotting factors IX, X, and XI, plasmin, and trypsin. Upon association with heparin, it inhibits blood clotting.

2.7. Cobalamin-binding Proteins 

(1) IF (standing for intrinsic factor) is a 114-kDa dimeric glycoprotein. It binds two molecules of vitamin B₁₂ (cobalamin) and helps its absorption in the intestine by binding to a specific receptor. (2) Transcobalamin I, II, and III are vitamin B₁₂-binding and transporting proteins in the bloodstream. I is a glycoprotein (carbohydrate content of 33% by weight) and has a molecular weight of 56,000. II is a simple protein of 60 kDa. (3) A protein known as R-binder (cobalophilin( also binds cobalamin.

2.8. Thyroid Hormone-binding Proteins 

The thyroid hormones, thyroxin (T4) and triiodothyronine (T3), are transported by thyroxin-binding pre-albumin, thyroxin-binding globulin, and serum albumin. Thyroxin and triiodothyronine in the blood are distributed among the three proteins in the ratios 70:10:20 and 40:30:30, respectively. 

1. Thyroxin-binding pre-albumin migrates on electrophoresis to the anodic side of serum albumin, has a molecular weight of 54,980, contains 0.4% sugar, and has four subunits. The serum concentration of this protein becomes low in patients with liver disorders.

2. Thyroxin-binding globulin is an acidic glycoprotein with a molecular weight of 60,000; it is present in serum at a concentration of 15 µg/ml and strongly binds thyroxin and triiodothyronine.

2.9. Steroid Hormone-binding Proteins 

These include corticosteroid-binding protein (transcortin), sex hormone-binding protein, and progesterone-binding protein. Transcortin is a glycoprotein, with a carbohydrate content of 16 to 26% by weight and molecular weight of 53,000.

 3. Serum b-Globulins

3.1.  Transferrin 

This is an iron-binding glycoprotein in the serum with a molecular weight of 77,000. When ferric ion is bound to transferrin, it will bind to the cell-membrane transferrin receptor of erythroblasts and reticulocytes and becomes internalized as a result of receptor-linked endocytosis; apotransferrin has low affinity for the receptor. The internalized transferrin releases the bound iron as the pH inside the endosome becomes lower, and then the receptor with bound apo-transferrin reassociates with the membrane, to recycle apo-transferrin into the serum. Transferrin has two iron-binding sites per molecule, but a similar protein found in hag-fish has only one iron binding site per molecule of 44 kDa. There are several genetic variants. A protein called conalbumin in egg white and lactoferrin in milk are actually apo-transferrin, with the same amino acid sequence as the transferrin in blood serum, but with different post-translational modifications . More than 60% of the iron used by blood-producing cells for the hemoglobin biosynthesis is supplied by transferrin.

3.2. b-Lipoproteins 

These include low-density and very-low-density lipoproteins . Serum lipoproteins are noncovalent complexes of specific proteins and lipids. They constitute a lipid transport system in the bloodstream and are classified in as chylomicrons, very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). They appear in the a-globulin fraction upon electrophoresis. A further subfraction, very-high-density lipoprotein (VHDL), is often identified. Each class of lipoproteins is characterized by specific apo-proteins and immunogenicities. The density classification of lipoproteins is based on lipid content; the higher the lipid content, the lower the density. The lipid content in each class of lipoproteins is not constant but varies within a certain range of values.

3.3. b₂-Microglobulin

 This is the smaller of the two polypeptide chains that constitute the histocompatibility complex antigen, but this protein does not have specific antigenicity. The amino acid sequence has a similarity to that of the constant domains of the immunoglobulin heavy chains.

3.4. Hemopexin 

This is a heme-binding protein with a molecular weight of 5700 and a carbohydrate content of 20%.

4. Serum g-Globulins

 Serum g-globulins constitute the secretory immune system of vertebrates and are classified asIgA, IgD, IgE, IgG, and IgM, with Ig standing for immunoglobulin. The basic structural unit of immunoglobulins may be seen in the H₂L ₂ structure of IgG, where H represents the heavy chain and L the light chain. Each light chain is disulfide-bonded to one of the heavy chains, and the two heavy chains are disulfide-bonded to form a covalently associated hetero-tetrameric subunit structure. Two antigen-binding sites are formed from the two pairs of H and L chains. The structural designs of other immunoglobulins are based on this IgG structure.

5. Egg-White Globulins

5.1. Ovoglobulins 

This fraction of egg white contains lysozyme and ovomacroglobulin as the major proteins in the G1 and G2 fractions, respectively. Lysozyme is a small basic protein with a molecular weight of 14,300 (129 amino acid residues), sedimentation coefficient of 1.9 S, and an isoelectric point of 11. It is an enzyme that cleaves cell-wall mucopolysaccharides at b(1–4) linkages between N-acetylmuramic acid and N-acetyl galactosamine, and thus has bactericidal activity. Chicken egg-white lysozyme is now called c-type lysozyme with a distinction from g-type lysozymes, such as that with 185 residues purified from goose eggs. Hen lysozyme constitutes about 3% of egg-white proteins and is readily crystallized. See also Lysozymes.

5.2. Ovomacroglobulin 

This is a large, tetrameric glycoprotein with a molecular weight of 700,000 (4 × 170,000). It has a proteinase-trapping activity similar to that of serum a2-macroglobulin. The two proteins are homologous in amino acid sequence and to the C3, C4, and C5 components of the blood clotting proteins. Unlike a₂-macroglobulin, ovomacroglobulin lacks the internal thiol ester bond. The presence of homologous proteins has been confirmed in the chicken, goose, crocodile, and turtle egg white (3).

6. Thyroid Globulins

 Thyroglobulin is a glycoprotein (10% sugar) of molecular weight 670,000, two identical subunits, sedimentation coefficient of 19 S, and pI of 4.5. It is synthesized as an iodine-free protein in the epithelium of the thyroid gland; later it is iodinated on some of its tyrosine residues by thyroid peroxidase. Two iodotyrosine residues dimerize to form the thyroid hormones 3,3′,5,5′-tetraiodothyronine (thyroxin) and 3,3′,5-triiodothyronine. Iodination also promotes the generation of covalent dimers of thyroglobulin through disulfide bond formation. Iodination of the protein is specific to the thyroid gland of vertebrates.

 7. Milk Globulins

 Cow's whey contains as the principal components (50 to 60%) beta-lactoglobulin, which is peculiar to ruminant milk, and alpha-lactalbumin. b-Lactoglobulin constitutes about 50% of the whey protein; it is fractionated as an albumin fraction that does not precipitate with 50% saturation ammonium sulfate, but the purified protein shows a globulinlike solubility in water. Whey protein is fractionated into three major fractions by centrifugation, and b-lactoglobulin is the major protein in the b fraction. It is a dimeric protein of 35 kDa, sedimentation coefficient of 2.8 S, and pI of 5.2. The lactoglobulin fraction includes b-lactoglobulin and immunoglobulin derived from the mother. The three-dimensional structure of this protein is similar to that of retinol-binding proteins (4).

References

 1. S. Cowan, M. E. Newcommer, and T. A. Jones (1990) Crystallographic refinement of serum retinol binding protein at 2 Å resolution, Proteins 8, 44–61. 

2. W. Borth, R. D. Feinman, S. L. Gonias, J. P. Quigley, and D. K. Strickland, eds. (1994). Biology of Macroglobulin. Its Receptor, and Related Proteins, Annals N. Y. Acad. Sci. 737. 

3. A. Ikai, M. Kikuchi, and M. Nishigai (1990) Internal structure of ovomacroglobulin, J. Biol. Chem . 256, 8280–8284. 

4. H. L. Monaco, G. Zonatti, P. Spadon, M. Bolognesi, L. Sawyer, and E. E. Eliopoulos (1987( Crystal structure of trigonalform of bovine -lactoglobulin and its complex with retinol at 2.5 Å resolution, J. Mol. Biol. 197, 695–706.