Water & pH

BIOMEDICAL IMPORTANCE
predominant chemical component of living organisms unique physical properties include: - ability to solvate a wide range of organic and inorganic molecules > derive from water's: - dipolar structure - exceptional capacity for forming hydrogen bonds - influences the structure of molecules excellent nucleophile slight propensity to dissociate into hydroxide ions and protons acidity of aqueous solutions is generally reported using the logarithmic pH scale

BIOMEDICAL IMPORTANCE
Bicarbonate and other buffers normally maintain the pH of extracellular fluid between 7.35 and 7.45 disturbances of acid-base balance are verified by measuring: - pH of arterial blood - CO2 content of venous blood Causes of acidosis (blood pH <7.35) include: - diabetic ketosis and lactic acidosis Causes of alkalosis (pH >7.45): - vomiting of acidic gastric contents regulation of water balance depends upon: - hypothalamic mechanisms that control thirst - antidiuretic hormone (ADH) - on retention or excretion of water by the kidneys, and on evaporative loss nephrogenic diabetes insipidus: - inability to concentrate urine or adjust to subtle changes in extracellular fluid osmolarity

WATER IS AN IDEAL BIOLOGIC SOLVENT

Water Molecules Form Dipoles Water Molecules Form Hydrogen Bonds

Water Molecules Form Dipoles

water molecule: - an irregular, slightly skewed tetrahedron - oxygen at its center two hydrogens and the unshared electrons of the remaining two sp 3-hybridized orbitals occupy the corners of the tetrahedron 105O angle between the hydrogens a dipole, a molecule with electrical charge distributed asymmetrically about its structure strongly electronegative oxygen atom pulls electrons away from the hydrogen nuclei while its two unshared electron pairs constitute a region of local negative charge

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Water
a strong dipole has a high dielectric constant dielectric constants: - for a vacuum is unity - for hexane it is 1.9; - for ethanol, 24.3 - water, 78.5 therefore it greatly decreases the force of attraction between charged and polar species relative to water-free environments with lower dielectric constantsstrong dipole and high dielectric constant: - enable water to dissolve large quantities of charged compounds such as salts

Water Molecules Form Hydrogen Bonds

Consequence: - hydrogen bonds favors the self-association of water molecules into ordered arrays: > hydrogen bonds influences the physical properties of water > accounts for its exceptionally high viscosity, surface tension, and boiling point > on average, each molecule in liquid water associates through hydrogen bonds with 3.5 others Hydrogen bonds are: - both relatively weak and transient - with a half-life of 1 usec or less - rupture in liquid water requires only 4.5 kcal/mol, < 5% of the energy required to rupture a covalent OH bond

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- enables water to dissolve many organic biomolecules that contain functional groups: > oxygen atoms of aldehydes > oxygen atoms of carbonyl carbons of ketones > nitrogen atoms of amides: - provide lone pairs of electrons that can serve as hydrogen acceptors Examples: alcohols and amines can serve both as hydrogen acceptors and as donors of unshielded hydrogen atoms for formation of hydrogen bonds

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INTERACTION WITH WATER INFLUENCES THE STRUCTURE OF BIOMOLECULES

Covalent & Noncovalent Bonds Stabilize Biologic Molecules Biomolecules Fold to Position Polar & Charged Groups on Their Surfaces Hydrophobic Interactions Electrostatic Interactions van der Waals Forces Multiple Forces Stabilize Biomolecules

Covalent & Noncovalent Bonds Stabilize Biologic Molecules

covalent bond: - the strongest force that holds molecules together noncovalent forces: - of lesser magnitude - make significant contributions to: > structure > stability > functional competence of macromolecules in living cells both can be either attractive or repulsive involve: - intramolecular interactions - intermolecular interactions - interaction with water

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Biomolecules Fold to Position Polar & Charged Groups on Their Surfaces

most biomolecules are amphipathic Examples: - proteins - lipids in bilogical membranes (phospholipid bilayer) Effects: - maximizes opportunities for formation of energetically favorable: > chargedipole > dipoledipole > hydrogen bonding interactions between polar groups on the biomolecule and water minimizes energetically unfavorable contacts between water and hydrophobic groups

Hydrophobic Interactions
refers to the tendency of nonpolar compounds to selfassociate in an aqueous environment: - driven neither by mutual attraction nor by what are sometimes incorrectly referred to as "hydrophobic bonds" - self-association minimizes energetically unfavorable interactions between nonpolar groups and water hydrogens of nonpolar groups such as the methylene groups of hydrocarbons do not form hydrogen bonds: - water molecules adjacent to a hydrophobic group are restricted in the number of orientations (degrees of freedom)

Electrostatic Interactions
interactions between charged groups help shape biomolecular structure termed salt bridges (electrostatic interactions between oppositely charged groups within or between biomolecules - salt bridges: > are comparable in strength to hydrogen bonds > act over larger distances > often facilitate the binding of charged molecules and ions to proteins and nucleic acids

van der Waals Forces

arise from attractions between transient dipoles generated by the rapid movement of electrons of all neutral atoms significantly weaker than hydrogen bonds potentially extremely numerous decrease as the sixth power of the distance separating atoms act over very short distances, typically 24

Multiple Forces Stabilize Biomolecules

DNA double helix illustrates the contribution of multiple forces to the structure of biomolecules: - each individual DNA strand held together by covalent bonds - the two strands of the helix are held together exclusively by noncovalent interactions: > hydrogen bonds between nucleotide bases (WatsonCrick base pairing) > van der Waals interactions between the stacked purine and pyrimidine bases helix presents the charged phosphate groups and polar ribose sugars of the backbone to water while burying the relatively hydrophobic nucleotide bases inside extended backbone maximizes the distance between negatively charged phosphates, minimizing unfavorable electrostatic interactions

H2O AN EXCELLENT NUCLEOPHILE

Many Metabolic Reactions Involve Group Transfer
Water Molecules Exhibit a Slight But Important Tendency to Dissociate

H2O AN EXCELLENT NUCLEOPHILE

Metabolic reactions often involve the process of nucleophilic attack Water, whose two lone pairs of sp3 electrons bear a partial negative charge, is an excellent nucleophile other nucleophiles of biologic importance include: - the oxygen atoms of: > phosphates > alcohols > carboxylic acids - sulfur of thiols - nitrogen of amines - imidazole ring of histidine

H2O AN EXCELLENT NUCLEOPHILE

Common electrophiles include: - carbonyl carbons in: > amides > esters > aldehydes > ketones - phosphorus atoms of phosphoesters Nucleophilic attack by water generally results in the cleavage of the amide, glycoside, or ester bonds that hold biopolymers together process is termed hydrolysis Conversely, dehydration: - monomer units are joined together to form biopolymers such as proteins or glycogen, water is a product, for example, during the formation of a peptide bond between two amino acids

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 hydrolysis is a thermodynamically favored reaction amide and phosphoester bonds of polypeptides and oligonucleotides are stable in the aqueous environment paradoxical behavior reflects the fact that the thermodynamics governing the equilibrium of a reaction dont determine the rate at which it will proceed enzymes accelerate the rate of hydrolytic reactions - proteases catalyze the hydrolysis of proteins into amino acids - nucleases catalyze the hydrolysis of the phosphoester bonds in DNA and RNA

Many Metabolic Reactions Involve Group Transfer

many of the enzymic reactions responsible for synthesis and breakdown of biomolecules involve : D-G + A <----> A-G + D Example: phosphorolytic cleavage of glycogen involve the transfer of glucosyl groups to water or to orthophosphate. equilibrium constant for the hydrolysis of covalent bonds strongly favors the formation of split products in many cases of biosynthesis of macromolecules thermodynamically unfavored formation of covalent bonds are involved enzymes in biological systems catalyze these reactions

Many Metabolic Reactions Involve Group Transfer

Two questions: 1. Given the nucleophilic character of water and its high concentration in cells, why are biopolymers such as proteins and DNA relatively stable? 2. And how can synthesis of biopolymers occur in an aqueous environment? central are the presenc of enzymes without enzymatic catalysis, reactions would proceed in a very slow pace regulation of enzyme activity is important as well as compartmentalization of these molecules

Water Molecules Dissociate

H2O + H2O <----> H3O+ + OH transferred proton is actually associated with a cluster of water molecules. protons exist in solution not only as H3O+, but also as multimers such as H5O2+ and H7O3+. the proton is nevertheless routinely represented as H+ 1 g of water contains 3.46 x 1022 molecules, the ionization of water can be described statistically for every hydrogen ion and hydroxide ion in pure water, there are 1.8 billion or 1.8 x 109 water molecules hydrogen ions and hydroxide ions nevertheless contribute significantly to the properties of water

 For dissociation of water,

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1 mole (mol) of water weighs 18 g, 1 liter (L) (1000 g) of water contains 1000 18 = 55.56 mol pure water is 55.56 molar since the probability that a hydrogen in pure water will exist as a hydrogen ion is 1.8 x 10 x-9, the molar concentration of H+ ions (or of OH- ions) in pure water is the product of the probability, 1.8 x 10-9, times the molar concentration of water, 55.56 mol/L The result is 1.0 x 10-7 mol/L

 We can now calculate K for pure water:

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The molar concentration of water, 55.56 mol/L, is too great to be significantly affected by dissociation. It therefore is considered to be essentially constant. This constant may therefore be incorporated into the dissociation constant K to provide a useful new constant Kw termed the ion product for water. The relationship between Kw and K is shown below:

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 Note that the dimensions of K are moles per liter and those of Kw are moles2 per liter2. As its name suggests, the ion product Kw is numerically equal to the product of the molar concentrations of H+ and OH-:
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At 25OC, Kw = (10-7)2, or 10-14 (mol/L)2. At temperatures below 25OC, Kw is somewhat less than 10-14, and at temperatures above 25OC it is somewhat greater than 10-14. Within the stated limitations of the effect of temperature, Kw equals 10-14 (mol/L)2 for all aqueous solutions, even solutions of acids or bases. We use Kw to calculate the pH of acidic and basic solutions.

pH IS THE NEGATIVE LOG OF THE HYDROGEN ION CONCENTRATION

The term pH was introduced in 1909 by Srensen, who defined pH as the negative log of the hydrogen ion concentration: This definition, while not rigorous, suffices for many biochemical purposes. To calculate the pH of a solution: 1. Calculate the hydrogen ion concentration [H+]. 2. Calculate the base 10 logarithm of [H+]. 3. pH is the negative of the value found in step 2.

pH = - log[H+]

 For example, for pure water at 25C,

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Low pH values correspond to high concentrations of H+ and high pH values correspond to low concentrations of H+. Acids are proton donors and bases are proton acceptors. Weak acids dissociate only partially in acidic solutions. Many biochemicals are weak acids. Exceptions include phosphorylated intermediates, whose phosphoryl group contains two dissociable protons, the first of which is strongly acidic.

Functional Groups that Are Weak Acids Have Great Physiologic Significance
functional groups that are weak acids or bases: - carboxyl groups - amino groups - phosphate esters knowledge of the dissociation of weak acids and bases thus is basic to understanding the influence of intracellular pH on structure and biologic activity charge-based separations such as electrophoresis and ion exchange chromatography also are best understood in terms of the dissociation behavior of functional groups

 We term the protonated species (eg, HA or RNH3+) the acid and the unprotonated species (eg, A or RNH2) its conjugate base. Similarly, we may refer to a base (eg, A or RNH2) and its conjugate acid (eg, HA or RNH3+). Representative weak acids (left), their conjugate bases (center), and pKa values (right) include the following:

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 We express the relative strengths of weak acids and bases in terms of their dissociation constants. Shown below are the expressions for the dissociation constant (Ka) for two representative weak acids, RCOOH and R NH3+.

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 Since the numeric values of Ka for weak acids are negative exponential numbers, we express Ka as pKa, where
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Note that pKa is related to Ka as pH is to [H+]. The stronger the acid, the lower is its pKa value. pKa is used to express the relative strengths of both acids and bases. For any weak acid, its conjugate is a strong base. Similarly, the conjugate of a strong base is a weak acid. The relative strengths of bases are expressed in terms of the pKa of their conjugate acids. For polyprotic compounds containing more than one dissociable proton, a numerical subscript is assigned to each dissociation in order of relative acidity. For a dissociation of the type
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the pKa is the pH at which the concentration of the acid R NH3+ equals that of the base RNH2.

From the above equations that relate Ka to [H+] and to the concentrations of undissociated acid and its conjugate base, when

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Thus, when the associated (protonated) and dissociated (conjugate base) species are present at equal concentrations, the prevailing hydrogen ion concentration [H+] is numerically equal to the dissociation constant, Ka. If the logarithms of both sides of the above equation are taken and both sides are multiplied by -1, the expressions would be as follows:

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Since -log Ka is defined as pKa, and -log [H+] defines pH, the equation may be rewritten as
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ie, the pKa of an acid group is the pH at which the protonated and unprotonated species are present at equal concentrations. The pKa for an acid may be determined by adding 0.5 equivalent of alkali per equivalent of acid. The resulting pH will equal the pKa of the acid.

HendersonHasselbalch Equation Describes the Behavior of Weak Acids & Buffers

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Solutions of Weak Acids & Their Salts Buffer Changes in pH

Solutions of weak acids or bases and their conjugates exhibit buffering, the ability to resist a change in pH following addition of strong acid or base. Since many metabolic reactions are accompanied by the release or uptake of protons, most intracellular reactions are buffered. Oxidative metabolism produces CO2, the anhydride of carbonic acid, which if not buffered would produce severe acidosis. Maintenance of a constant pH involves buffering by phosphate, bicarbonate, and proteins, which accept or release protons to resist a change in pH. The value of pKa relative to the desired pH is the major determinant of which buffer is selected. The solution resists changes in pH most effectively at pH values close to the pKa . A solution of a weak acid and its conjugate base buffers most effectively in the pH range pKa = +/- 1.0 pH unit.

Acid Strength Depends on Molecular Structure

Many acids of biologic interest possess more than one dissociating group. The presence of adjacent negative charge hinders the release of a proton from a nearby group, raising its pKa. This is apparent from the pKa values for the three dissociating groups of phosphoric acid and citric acid. The effect of adjacent charge decreases with distance. The second pKa for succinic acid, which has two methylene groups between its carboxyl groups, is 5.6, whereas the second pKa for glutaric acid, which has one additional methylene group, is 5.4.

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pK a Values Depend on the Properties of the Medium

profoundly influenced by the surrounding medium: - medium may either raise or lower the pKa effect of dielectric constant on pKa: - pKa of a carboxylic acid increases -pKa of an amine decreases because ethanol decreases the ability of water to solvate a charged species pKa values of dissociating groups in the interiors of proteins thus are profoundly affected by their local environment, including the presence or absence of water