Which of these is a carboxyl group?

2.4 Carboxyl Groups

Carboxyl groups are a combination of two functional groups attached to a single carbon atom, namely, hydroxyl (

Unlike the various functional groups discussed above, some studies have reported the preferential differentiation of hMSCs toward a chondrogenic lineage in the presence of carboxyl-modified surfaces.7 One potential reason may be that carboxyl groups are the predominantly exposed functional groups of native cartilage, which is composed of glycosaminoglycans. Curran et al. incorporated carboxyl functional groups on silane-modified glass surfaces and showed that carboxyl-coated surfaces induced chondrogenic differentiation of hMSCs in both basal and chondrogenic media. The modified surface maintained highest viable cell adhesion under chondrogenic conditions and showed early production of type II collagen, demonstrating the suitability of the substrate for the chondrogenic differentiation of hMSC.

This was further supported by another study wherein carboxyl group-modified PEG hydrogels were used as 2D and 3D substrates to culture hMSCs.18 This study showed that hMSCs cultured on surfaces with high concentrations of carboxylic acid exhibit upregulation of collagen type II. Moreover, fluorescence in situ hybridization analysis detected increased expression of aggrecan (cartilage-specific protein) after 10 days of culture on carboxyl-modified surfaces. Morphological findings supported the conclusion, with cells exhibiting a characteristic round morphology of chondrocytes on the acid-modified surfaces.

9.13.3.4 Carboxyl Group Reduction

Carboxyl group reduction of the CPS and LPS samples was performed as previously described.55 Briefly, LPS (10 mg) was dissolved in distilled water (10 ml) and following the addition of 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (113 mg), the stirred mixture was maintained at pH 4.7 by titration with 0.1 mol l−1 HCl for 3 h. Following completion of the reaction a 2 mol l−1 solution of sodium borohydride (12.5 ml) was added slowly and the reaction mixture was maintained at pH 7 by titration with 4 mol l−1 HCl. The reaction was allowed to proceed for 2 h at 22 °C, and the solution was dialyzed and lyophilized. The product was purified by gel permeation chromatography on Sephadex G-100 and lyophilized (yield 6 mg).

Effect of Buffering Power of Proteins on Carbon Dioxide Carriage

Amino and carboxyl groups concerned in peptide linkages have no buffering power. Neither have most side chain groups (e.g. in lysine and glutamic acid) because their pK values are far removed from the physiological range of pH. In contrast is the imidazole group of the amino acid histidine, which is nearly the only amino acid to be an effective buffer in the normal range of pH. Imidazole groups constitute the major part of the considerable buffering power of haemoglobin, with each tetramer containing 38 histidine residues. The buffering power of plasma proteins is less and is directly proportional to their histidine content.

The four haem groups of a molecule of haemoglobin are attached to the corresponding four amino acid chains at one of the histidine residues on each chain (page 178), and the dissociation constant of the imidazole groups of these four histidine residues is strongly influenced by the state of oxygenation of the haem. Reduction causes the corresponding imidazole group to become more basic. The converse is also true: in the acidic form of the imidazole group of the histidine, the strength of the oxygen bond is weakened. Each reaction is of great physiological interest and both effects were noticed many decades before their mechanisms were elucidated.

The reduction of haemoglobin causes it to become more basic. This results in increased carriage of carbon dioxide as bicarbonate, because hydrogen ions are removed, per­mitting increased dissociation of carbonic acid (first dissociation of Equation 3). This accounts for part of the Haldane effect, the other and greater part is due to increased carbamino carriage (see earlier discussion).

Conversion to the basic form of histidine causes increased affinity of the corresponding haem group for oxygen. This is, in part, the cause of the Bohr effect (page 178).

Total deoxygenation of the haemoglobin in blood would raise the pH by about 0.03 if the Pco2 were held constant at 5.3 kPa (40 mm Hg), and this would correspond roughly to the addition of 3 mmol of base to 1 litre of blood. The normal degree of desaturation in the course of the change from arterial to venous blood is approximately 25%, corresponding to a pH increase of about 0.007 if Pco2 remains constant. In fact, Pco2 rises by approximately 0.8 kPa (6 mm Hg), which would cause a decrease of pH of 0.040 if the oxygen saturation were to remain the same. The combination of an increase of Pco2 of 0.8 kPa and a decrease of saturation of 25% thus results in a fall of pH of 0.033 (Table 9.2).

Ionic Groups

Compounds that contain carboxyl groups are called acids (or carboxylic acids). As illustrated in Figure 1.1, a carboxyl group in aqueous solution is partially ionized to the carboxylate anion. The degree of ionization depends on the dissociation constant of the acid and the initial pH of the solution. The strength of these acids varies somewhat depending on the attached R group. Esters are formed by reaction of a carboxylic acid with an alcohol. Amides are formed by reaction of a carboxylic acid with an amine.

Inorganic phosphate and organic phosphates are ionized when dissolved in water. Similarly, inorganic sulfate and organic sulfate are ionized when dissolved in water. In inorganic phosphate and sulfate, the R group is a hydrogen atom.

As also illustrated in Figure 1.1, a primary or secondary amine group can function as a weak base. The degree to which the group is protonated to the positive ion depends on the dissociation constant of the molecule to which it is attached and on the initial pH of the solution.

7.2 Crystal structures and physical properties of flurbiprofen salts

Flurbiprofen is bearing a carboxyl group. As the free acid its aqueous solubility is only 0.03 mg/mL. Hydrophobicity of the counter ion does not fully determine the solubility of its amine salts, being 0.37, 2.80, 0.64, and 0.17 mg/mL for the cyclohexyl (CH)-, hexyl-, octyl-, and adamantyl (AD)-ammonium salts, respectively. ΔH of fusion is 159.0 J/g for the CH but only 81.0 J/g for the hexylammonium salt. It was reported that the structures of the stable CH and AD salts, acquired with synchrotron radiation because they exist as fine needles [79].

In both cases, the cycloalkyl group covers the twofold disordered fluorophenyl ring, forming a clear hydrophobic domain. Hydrogen bonds join three ammonium H atoms to two carboxylate O atoms and create infinite ladders along the short b-axis, which in CH shows no thermal expansion, while the a-axis expands by 1.9% over 141 K (Table 4.4) [80,81].

Table 4.4. Crystal data of flurbiprofen salts [80,81]

133.3 Monounsaturated Fatty Acids and GLP-1

Monounsaturated fatty acids possessing a free carboxyl group indeed stimulate the secretion of intestinal proglucagon-derived peptides, including GLP-1, from fetal rat intestinal cultures, this effect being lost upon full saturation of the concerned fatty acids (Rocca and Brubaker, 1995). Various studies deal specifically with the effects of monounsaturated fatty acid diets on GLP-1 secretion and glycemic tolerance. Thomsen et al. (1999) first compared the postprandial responses of glucose, insulin, fatty acids, triacylglycerol, gastric inhibitory peptide (GIP), and also that of GLP-1, to test meals rich in saturated and monounsaturated fatty acids, in young, lean, healthy subjects. GLP-1 and GIP responses were higher after ingestion of a meal containing 80 g olive oil than those after either 50 g carbohydrate (control meal) or 100 g butter (saturated fatty acid meal); this coincided with a lower average of blood D-glucose concentrations after the olive oil meal than after the control meal; however, the early peak of blood D-glucose and plasma insulin concentrations were highest after ingestion of the control meal, and no significant differences in glucose, insulin, or fatty acid response to the two fat-rich meals were seen (Table 133.3). In a comparable study later conducted by the same investigators in overweight patients with type 2 diabetes (Table 133.3), the GLP-1 response was again highest after the olive oil meal, while no significant difference was seen in the glucose, insulin and fatty acid responses to the two fat-rich meals (Thomsen et al., 2003).

Table 133.3. Responses to a test meal rich in monounsaturated fatty acids compared to a saturated fatty acid rich meal.

This table shows the qualitative changes of plasma glucose and lipids, and of the secretion of insulin, GLP-1 and GIP (gastric inhibitory peptide) in healthy and obese type 2 diabetic subjects fed on a monounsaturated fatty acid rich meal (80 g olive oil) compared to a saturated fatty acid rich meal (100 g butter).

=: no change; ↑: significantly higher; ↓: significantly lower.

In lean Zucker rats (Rocca et al., 2001), pair-fed for 2 weeks with a synthetic diet containing 5% fat derived from either olive oil (74% monounsaturated fatty acids) or coconut oil (87% saturated fatty acids), the olive oil-fed rats showed improved glucose tolerance, compared with the CO group, to both oral and duodenal glucose. Despite such a difference, the plasma insulin pattern was comparable in the two groups, but the secretion of gut glucagon-like immunoreactive material during the duodenal glucose tolerance test was higher (min 10) in the olive oil-fed rats than in coconut oil-fed rats. The benefit in glycemia tolerance conferred by feeding was abolished when the GLP-1 antagonist exendin9–39 (Figure 133.3) was infused 3 min before the duodenal glucose administration.

Loss of Heparan Sulfate Proteoglycan Charge Barrier

HSPGs, with multiple sulfate and carboxyl groups on their glycosaminoglycan side chains, are strongly negatively charged. The loss of HSPG integrity may reduce the charge barrier imparted by the GBM and increase glomerular permselectivity (Fig. 21-4). Injury to HSPG may also impair podocyte and glomerular endothelial cell binding, and may release HSPG-bound growth factors (FGF, VEGF, heparin-binding epidermal growth factor–like growth factor).92 Importantly, agents that injure HSPG in the GBM may also affect HSPG in the endothelial glycocalyx.

A reduction in GBM anionic sites or decreased heparan sulfate staining have been described in several animal models of glomerulonephritis.93–98 It has also been described in human glomerular disease.99,100 In some, the degree of heparan sulfate staining has been inversely correlated with albuminuria.97,99–101 In diabetic nephropathy, functional studies have demonstrated an increase in pore size and decrease in charge barrier.47,49,102 These functional changes have been correlated with a decrease in heparan sulfate staining, mostly without a reduction in HSPG core protein.99,101 Both decreased synthesis of heparan sulfate (35S-sulfate incorporation)103,104 and structural modification (undersulfation) have been described.104

Injury to the heparan sulfate components of the GBM may be caused by several mechanisms, including oxidative stress and enzymatic digestion (neutral serine proteases, heparanases). Reactive oxygen species (ROS) may be formed in the kidney from resident or infiltrating cells105 and play a central pathogenic role in several models of proteinuria, including passive Heymann nephritis,106 adriamycin nephropathy,107,108 and puromycin aminonucleoside nephropathy.109,110 Although ROS may directly cause podocyte injury, or can be derived from podocytes that are injured, there is also evidence that ROS may promote degradation of heparan sulfate. In vitro, heparan sulfate side chains of agrin may be depolymerized by ROS.100 Peroxynitrite, derived from nitric oxide, can degrade hyaluronic acid and possibly other glycosaminoglycans.111 In adriamycin nephropathy, albuminuria correlates with the decreased heparan sulfate staining, and both are ameliorated by the antioxidant dimethylthiourea.100

Enzymatic cleavage of HS side chains may also be an important factor in the development of proteinuria.93,112 Neutral serine proteinases (elastase, cathepsin G) are released by activated polymorphonuclear cells. In vitro, cationic elastase binds to anionic heparan sulfate.113 Renal infusion of these cationic enzymes results in the degradation of heparan sulfate and is associated with marked proteinuria, notably without foot process fusion seen on electron microscopy.114 There was also no change in the other structural GBM components (type IV collagen, laminin, fibronectin).115 In murine anti-GBM nephritis, proteinuria is ameliorated by elastase inhibitors116 or abrogated in beige mice, which are deficient in elastase and cathepsin G.117

Heparanase, an endoglycosidase specific for heparan sulfate, is released from activated polymorphonuclear cells and platelets, and has been shown to degrade heparan sulfate in extracellular matrix.118 Transgenic animals that overexpress heparanase develop spontaneous proteinuria associated with foot process effacement and renal impairment.119 Heparanase expression in the podocyte (and glomerular endothelial cell) is upregulated in several models of proteinuria.120,121 Blocking heparanase activity with an anti-heparanase antibody121 or synthetic inhibitor PI-88122 has been shown to ameliorate proteinuria in passive Heymann nephritis without changing the binding of antibody or C5b-9.

By contrast, although perlecan knockout mice die during embryonic development, mice with mutant perlecan leading to the loss of heparan sulfate side chains do not develop hematuria or proteinuria and do not have GBM structural abnormalities.123 However, other proteoglycans such as agrin compensate for this loss. Agrin-deficient mice develop abnormalities at the neuromuscular junction, but GBM abnormalities have not been studied.124

Primary Structure

The primary structure of a protein is simply the linear sequence of amino acids held together by peptide bonds. The higher orders of structure, including any disulfide bonds, are determined in part by the primary structure. Since the primary structure correlates directly with the sequence of triplet bases in the corresponding gene, the genetic code contains a specification for all levels of protein structure.

The linear sequence of amino acids is read from left to right, with the amino terminal on the left. The tetrapeptide below is called alanylaspartylglycylleucine:

+H3N-ala-asp-gly-leu-COO− or +H3N-Ala-Asp-Gly-Leu-COO−

Peptide bonds are amide bonds between the α-carboxyl group of one amino acid and the α-amino group of another (Fig. 3-1). The result is a planar structure that is stabilized by resonance between the α-carboxyl and α-amino groups. The side chains are able to extend out from the peptide chain and interact with each other or with other molecules.

PATHOLOGY

The polymerization of amino acids produces a linear molecule referred to generically as a polypeptide (Fig. 3-2). More specific nomenclature can indicate the number of amino acids in the polypeptide, e.g., dipeptide (two amino acids) or oligopeptide (relatively few amino acids). The properties of a polypeptide are determined by the side chains of their amino acids.

Peptide Bond

Amino acids can establish covalent bonds between the carboxyl group of one amino acid and the α amine group of another. This amide type link is called a peptide bond and it is accompanied by the loss of water (Fig. 3.7).

The product formed by linking two amino acids together is called a dipeptide. The subsequent binding of additional amino acid units to this dipeptide via peptide bonding generates tripeptides, tetrapeptides, pentapeptides, etc. The polymers formed by more than 10 amino acids linked by peptide bonds are designated polypeptides. A polypeptide chain is considered a protein when it has a molecular mass greater than 6000 Da (Daltons). Dalton (Da) is the unit of atomic mass; it is 1/12 the mass of one atom of 12C. Frequently the expression kilodalton (kDa), 1000 daltons is used. Relative mass is the ratio between the molecular mass of a given substance and the mass of one atom of 12C, which corresponds to the mass of a polymer of more than 50 amino acids. Below this mass, the compounds are designated peptides. There is no precise distinction between peptides and proteins; 6000 Da is arbitrary and was chosen because it is the approximate mass of insulin, a hormone produced in the pancreas, which was the first protein whose entire structure was deciphered.

At one end of every polypeptide chain there is an amino acid with a free α amine group. By convention, this end is considered the beginning of the chain and is called the amino-terminal or N-terminal portion of the polypeptide. The other extreme of the chain ends with a free carboxyl group and it is considered the C-terminal end of the polypeptide chain.

When integrated in the peptide or protein chain, amino acids lose the H of the amine group and the OH of the carboxyl group that are involved in the peptide binding. The amino acid units forming the polymer are referred to as amino acid residues.

4.4.1.2.1 Chemicals

(1)

1–1.25 nmol NMs with carboxyl groups (QD–COOH) on the surface in 100–125 µL DI water.

2 mg of EDC in 0.5 mL buffer A.

1 mg NHS (sulfo-N-hydroxysuccinimide).

Buffer A: 1.5 mL of 0.01 M H3BO3, pH 5.5.

Buffer B: 2 mL of 0.01 M H3BO3, pH 8.5.

Buffer C: 0.1 mL of 1 M glycine or 1 M lysine.

Buffer D: 3 mL 0.01 M of 0.01 M H3BO3, pH 7.2.

Biomolecules (protein, DNA, etc.) in 0.01 M H3BO3 (or PBS), pH 7.0–7.4. If the biomolecule is in a different buffer and/or contains glycerol, these must be removed through dialysis, ultracentrifugation, or spin filtration to replace the buffer with H3BO3.