Introductory Concepts 560D on-line Updated

8/3/2019 Introductory Concepts 560D on-line Updated

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Introductory ConceptsFor Biochemistry 560BDr. Charles Saladino

In this course, we shall be covering material that is contained within

many texts and other reference sources. However, as your professor, Ibelieve in the strongest of terms that my role should not only be topresent to you biochemical principles and specifics, but to show youhow to learn this material through understanding, conceptualizing,extrapolating, making correlations within it, so that you will be able torelate it to clinical sequelae with these thought processes continuingthroughout this course. In other words, we need to memorize, but wealso need to appreciate cellular biochemistry by understanding what itis doing and what biomolecular strategy is being used in the process.In this regard, besides book material, you shall feel my own touch uponthis course. This means I have had to prioritize material for which

years worth of courses can be taken. Thus, in practical terms, I amforced to decide what should be included and what can not be covered,due to time constraints, as I so gladly share with you the biochemical,cellular, and organismal context within which this awesome andbasically miraculous interplay of cellular chemistry occurs. If youchoose to take advantage of this approach to learning, you could reallyappreciate and perhaps even love a course which might otherwiseintimidate some. Enjoy this experience, and hopefully, we will beexploring an incredible world together.

We can begin with three major concepts about which I have thought

long and hard and which guide a major portion of my approach tobiochemistry. Even though I might not refer to these concepts duringa given lecture, please keep them in mind as we explore varioustopics. When possible, try to apply them to whatever subject wediscuss.

The first concept is relatively simple. I refer to it as thestructure/function relationship. That is, each cellular component is sostructured as to function most efficiently. That seems quite obviousand is a simple concept when you think, for example, of a bone notfunctioning properly when it is broken. However, when you consider

cell structure, then it becomes more detailed. If we look at a cellmembrane (and we will later on in the course) composed, in part, ofvarious phospholipids, that membrane will have dynamic fluidityproperties that are dependent, again in part, upon the fatty acidcomposition of those phospholipids. Thus, if we alter the fatty acidcomposition of the membrane (such as occurs when a hyperlipidemicstate alters the composition of platelet membranes surprised?), thenwe can expect the membrane to have a change in its biophysical


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properties, including a different fluidity within the membrane structure.So why is this important? With receptors traversing the membranefrom the outside of the cell into the cytoplasm, one could expectalterations of at least the transmembrane portion of that receptor, witha subsequent change in receptor function and subsequent cellular

transignaling. This could ultimately affect specific metabolic pathwayswithin that cell. Before the course is completed, I will hopefully havetime to elaborate on this, perhaps by talking about some of theresearch I have done in this area. However, in the mean time, I will askyou to keep this important structure/function concept in mindthroughout the course. This particularly applies to what I hope is someof your knowledge about topics such as the function-based threedimensional structure of proteins (especially enzymes), as well as tothe electron transport system and the proton gradient that isgenerated along the inner mitochondrial membrane during oxidativephosphorylation (ATP production). Let me give a specific, heads-up

examples regarding this topic:

The first example refers to proteins that are embedded into the innermitochondrial membrane at a precise distance apart that allows for theefficient transfer of electrons. This electron transport, as we shall seein a later lecture, generates the energy needed to drive protons acrossthe space between the inner and outer mitochondrial membranespace. The protomotive force ultimately generates the energyrequired to form ATP. If a drug is used to expand the entiremitochondrion, then the inner membrane is stretched, the proteins arenot at the proper distance for electron transfer, and ultimately ATP can

not be generated.

The other example I wish to use illustrates the structure/functionconcept at the most fundamental level known in the universe - that is,the quantum/electron level. So, during digestion, the enzyme lactasesplits the sugar lactose into its components - glucose and galactose. Inorder for this to be accomplished, the lactase must fire a proton intothe heart of the lactose. Now this particular proton is usually bondedto a key, obviously electronegative, oxygen. But sometimes, theproton jumps to a neighboring nitrogen (also electronegative -although less so than oxygen). The proton can only be fired into the

lactose when it is on the oxygen. So, how does the enzyme know thatthe proton is on the oxygen and not the nitrogen????? Is this notincredibly amazing!!!

The second concept is a bit more complicated, because it requireseven more of an understanding of subcellular and molecular inter-


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relationships than does the first principle with which we began ourdiscussion. Let us refer to this second concept ascompartmentaliziation. At the cellular level, we might define this as alocalization of certain biochemical sequences which are oftenphysically separated from other biomolecular reactions by highly

hydrophobic membranes that help form specific structural barrierswithin various portions of the cell. Just stop for a moment, andconsider this concept carefully. What does it really mean, and,therefore, what does it really imply? The answer is a great deal. Letus start here with some illustrative examples.

We all remember from our most elementary courses that themitochondria is where the great majority of the ATP is produced in cellsthat in fact contain those structures. Thus, a specific electrochemicalenvironment is required for this ATP production, one that is quitedifferent from that which is found in most other parts of the cell. Such

an environment would not be particularly conducive to other chemicalreactions that must take place in the free cytoplasm, for example. Theelectrochemical, oxidative/reductive environment is closely related tolocal pH, which is a critical factor in the way a protein folds, as well asin providing the proper environment to support a myriad of oxidationvs. reduction reactions. Thus, compartmentalization, therefore, allowsfor specific intracellular microenvironments that enhance the efficiencyof specific biochemical pathways upon which a viable cell mustdepend.

Another important point by way of example: There are numerous

regulatory steps and specific molecules which govern the formation ofATP in the mitochondria. One of many is the availability of phosphateion, as one would expect. The phosphate ion must cross into themitochondria to be available for oxidative phosphorylation. Bycompartmentalizing the bulk of ATP formation to the mitochondria,easier and more efficient regulation of that process can occur by (inpart) controlling access of critical components (like phosphate ion) tothe inner mitochondrial membrane, which is where ADP is convertedinto ATP. Further, responding to the need for this high energycompound in other parts of the cell, egress of ATP from themitochondria can be regulated more easily.

One more example: The cellular lysosome is a single-membranebound structure which contains a large spectrum of hydrolyticenzymes used to break down various biomolecules, whose componentsare likely to be recycled etc. If those enzymes were allowed to beactivated without being compartmentalized, severe cellular damagecould result in a random manner, as in fact does occur under aberrantconditions. This is but one of many examples of isolating reactions that


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can not be mixed into an environment with other reactions.

Before closing this topic, I would like to ask you a few rhetoricalquestions very much related to compartmentalization. If I were to askyou why we arent one big ameba, you might laugh and say you

actually think you know someone who is! However, seriously, what Iam really asking you is why we arent one giant cell? Why are we soterribly multicellular? I am sure many of you would say, Oh, cellspecialization, or perhaps, It is more efficient. Those would be truestatements, but do they really get at the heart of compartmentalizingvarious bodily functions to specific cells and thus tissues and organs?Not really. After all, one could argue that an ameba or parameciumhas some type of neural-like response, feeds, digests, has sex, etc. Ofcourse I am certainly not implying that we are no better off than asingle cell organism, especially with our wonderful brain and thefantastic mind that emanates from that incredible tissue. So where am

I going with all this?Ask yourself how long it would take an oxygen molecule to diffuse fromthe outside of your nose to where your heart would be, if we were onebig cell. Ask yourself how long it would take to transignal a metabolicstimulus from a receptor on the surface of your skin to a metabolicreaction located somewhere in the middle of you body? The answersnow become obvious. It could never happen that way. Why?Contemplate this. This should really get you thinking. Might youconsider surface area to volume ratios? Think about how much greatera surface area a hummingbird has than an elephant when compared to

their respective volumes. That ratio is much greater in ahummingbird, and I ask you which has the higher metabolic rate? Whohas a greater metabolic rate and has less problem keeping off weight(all other things being equal), a 65 250 lb person, or one who is 45and 250 lb? Obviously, the answer is the person with a high surfacearea to volume ratio. Make sense?

Thus, in the mean time and in summary, compartmentalizationprovides for greater cellular control of its own biochemistry than wouldotherwise be the case, and it increases the surface area to volumeratio to provide more surface area upon and in which reactions can

occur. Of great importance is the fact that this translates into greaterefficiency with which cellular function can take place.

At the heart of the third concept to which I wish to introduce you is thisextremely important term efficiency. Unless otherwise stated, youcan correctly assume the word efficiency to mean energy beingutilized in the cell in the most optimal physiochemical manner possible.In other words, the cell has been so designed so as to waste as little


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energy as possible, understanding that no machine - physical orchemical - is 100% efficient.

The use and production of cellular energy is governed by the samelaws of the universe as are the stars, a waterfall, or a chemical reaction

in a test tube. For us, the law most relevant to cellular metabolism isthe Second Law of Thermodynamics, with which I hope you have somefamiliarity. We absolutely must appreciate the relationship of its termsto each other, if we are understand why we really need to consumeparticular nutrients, how energy-requiring metabolic reactions are ableto occur, and what happens to some of the energy derived from thosereactions that produce it. This inter-relationship is absolutely critical tomaintaining a viable living system. So lets start.

G = H TS

As I am sure you already know, the symbol refers to a change infor all terms to which it is attached. Thus:

G = free energy change the energy available to dowork

H = enthalpy or heat content changeT = temperature in degrees KelvinS = entropy (disorder, randomness, chaos) change

If we look at the overall equation and try to summarize it, we shouldconclude that the natural tendency of the universe is to move towardrandomness or disorder (a more + S term). That applies to

macromolecules, cellular structure, and the whole organism.

How fast entropy (disorder, chaos, randomness) is approached andultimately achieved can be directly modulated by the temperature andhow much free energy is available. We can dispense with the T in thiscourse, because we can not substantially alter our body temperature(in degrees Kelvin) sufficiently enough affect a change in entropy, aswe certainly could do in a test tube reaction to which heat is easilyadded. Simply stated, we are warm-blooded organisms.

I will not spend much time on the H term either, which represents the

difference in heat content of reactants vs. products of a reaction.However, please remember that an endothermic reaction (where H ispositive) requires energy to make that reaction go. On the other hand,an exothermic reaction (where H is negative) produces heat. Of thatheat produced, some is dissipated and is absorbed by the watercontained in the cell, contributing to our constant body temperature(because water has a high specific heat). The rest of that exothermicenergy can be utilized to do work. This useful energy (that which is


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available to do work) is known as the free energy (the G term) of areaction and is a form of energy upon which countless, life-sustainingbiochemical reactions rely, in order to go to completion.

It is now very important to our understanding of metabolism that we

carefully look at this G term. With the appreciation that the value ofG is dependent upon all the other terms in the Second Law Equationshown above, we must know whether that G term is positive ornegative. This is absolutely critical to understanding metabolism andis not just a theoretical exercise that requires arithmetic. In brief, ifthe G term is positive, then the reaction is said to endergonic and willnot occur spontaneously. In significant contradistinction, a reaction issaid to be exergonic, and thus spontaneous, when the G value isnegative. In chemistry we define spontaneous a little differently thanone might use it in straight vocabulary. Spontaneous means that areaction might need a little energy push to get started; but once it

starts going, it needs no further outside influence to go to completion.Conversely, endergonic reactions must have a continual source ofenergy input, in order to complete the reaction. We will investigatethis in much more detail when we discuss enzyme kinetics, as well asenergy biotransformation. For now, however, let us clearly delineatethe idea that anabolic reactions (where biomolecules are synthesized)are most likely to be endergonic, whereas catabolic reactions (wherebiomolecules are broken down into simpler components) are probablygoing to be exergonic.

Again, I give you this background not as a theoretical exercise. Rather,

we need to appreciate the reason why, for example, the Krebs cycle(which we certainly will cover in this course), as it passes through eachof its steps, is never reversible as a whole cycle. The answer is thatthe direction the Krebs cycle takes is because the cycle as a whole hasa large negative G. In fact, almost every step has a negative Gvalue. Reversing the Krebs cycle would not be spontaneous, and thuswould require energy (and the enzymes) that would simply not beavailable. This is no accident.

We can close this major (third) concept of the Second Law by pointingout that ultimately, entropy wins out, and individual cells, and finally

the whole organism, dies. This represents disorder, compared tovibrant living systems, which display more order (less randomness orless disorder). The higher the negative S value, the greater chanceorder or structure will need to be maintained by an imput of freeenergy from somewhere. So the chaos or disorder term relies uponthe value of the G term. In other words, this does not occur in avacuum. To modulate or slow down the disorder term in a living cell,the Second Law tells us that free energy must be available to do the


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work of synthesis and the maintenance of cell structure and function.Take the extreme case of starvation. Little by little, without a renewedenergy input (negative G), the cells and the organism approachgreater and greater positive S (randomness, disorder, chaos), and theorganism dies. Thus, what better justification for requiring

nutrition????? Of course what we eat is another matter.

A final personal note: I have written this Introduction spontaneouslyand without looking at any reference material. Now I am certainly notsaying this so as to pat myself on the back hardly. Rather, my pointis that I consider the above material so fundamental to understandingmany of the whys of biochemistry, that I carry these thoughts aroundwith me whenever I consider human metabolism and nutrition.Therefore, I am saying to you, make sure you fully grasp what I havejust written here. Keep it with you; and attempt to apply theseprinciples as we discuss the biochemistry of human nutrition.

Last, but not least: Please dont allow yourself to fall into the quicksandof thinking that, I will never use this in my practice. If you do, thenyou will miss the opportunity to critically analyze a great deal ofimportant future data presented in the scientific literature. Believeme, if you grasp these underlying principles of metabolism, you will bea better health care provider, because you will think critically at ahigher level. In other words, I would simply offer to you the thoughtthat one should strive not to be a technician only in their practice, andthat the empirical approach to reading the biomedical literature and totreating a patient is not the superior one, when compared to having

the understanding of and appreciation for the depth of the cellular andmolecular inter-relationships that are biochemistry. So letscontinue..

The Aqueous Environment

We all know that the living cell contains a very large percentage ofwater by weight, depending upon the cell, of course; and this is alsothe case in the various extracellular compartments. Although adiscussion of the details of the many roles of water in terms of fluidbalance, osmotic considerations, etc. is not appropriate for this course,

we do need to consider the aqueous compartment of cells because ofthe fact that water is a polar molecule (with oxygen being moreelectrophilic than hydrogen, finalizing a relatively positive change atthe hydrogen atoms and a relatively negative charge at the oxygen).Obviously then water is very cohesive, because it hydrogen bonds withother water molecules. It would then follow that such cohesivenesswould dramatically affect interactions between molecules in solution.Specifically, the polarity and hydrogen bonding capability of water


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make it highly interactive, in that water is an excellent solvent for polarmolecules. The reason for this is that water weakens the electrostaticand hydrogen bonding between polar molecules (including ions) bycompeting for their attractions, which is why non-polar molecules arewater-insoluble.

What does this mean in practical terms? I would think the existence oflife depends critically on the capacity of water to dissolve a remarkablearray of molecules that include fuels, building blocks, catalysts, andcarriers of information. Point: This means that high concentrations ofthese polar molecules can coexist at the same time in the waterenvironment, where they can diffuse around and interact with oneanother.

You might want to ask this question, if you are doing some realthinking. Because water would appear then to weaken interactions

between polar molecules, wouldnt this cause a problem? Well,another of what will be many examples of a truly incredible design isthat many proteins can create a water-free environment within aprotein (yes a microenvironment within a molecule contemplatethat), which would allow for hydrophobic interactions within the proteinto occur. Thus, the posed problem is circumvented. We will see moreof this in detail when we study protein structure. We will also see lateron that water actually helps drive the folding of proteins by helpingwith favorable energy changes.

Now, what I am about to discuss might seem a bit overwhelming and

unrelated to your future as a clinician in terms of knowledge that youneed. However, I am presenting the following to start to develop morecontemplative thinking skills, and to give you a better appreciation ofhow very, very complex the topic of even the simplest ions can bewhen it comes to the aqueous environment. So here goes:

Now when we say the aqueous environment, we have a tendency toautomatically think of water as it would come out of kitchen faucet. Tobe sure, some of the cellular water does indeed take this form with anorderly hydrogen bonding mediating the organization of what we willcall bulk water - that is water as we first think of it. However, water

can take on an organization that is more ordered and more ice-like.Obviously there is no ice in the cytosol of the cell, but water undercertain conditions is found to be more hydrogen bonded than bulkwater, although not as much as what we would observe in ice.

This should begin to spark your thinking, because if our very polarwater is organized (hydrogen bonded to itself) to different degrees indifferent parts of the cytoplasm (which is the case), then it would


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follow that the various forms of water create certainmicroenvironments. It would also follow that ions like sodium andpotassium would not be distributed homogeneously throughout the cellcytosol and, thus, neither would the electrochemical environment. Letme explain that a bit.

Think about a cellular protein with its surface charges arising from thevarious amino acid carbonyl (-) and amino (+) groups and the R groups(side chains) (+ or -). Further, you would expect each amino acidcomprising the surface of the protein to present several charges.These charges would have a substantial effect upon water, which beinga dipole, would orient according to the surface charge on the protein.Thus a layer of what we are now calling vicinal water will line up alongthe proteins charged surface - say the positive side of water to thenegative charge on the protein (or vise versa). In addition, because ofthe dipolar nature of water, we can expect layers of water to form

along at least some of the surface of proteins, with the layering limitedby the distance of the water from the charged protein surface.

The implications of all this are quite staggering. Think about it. Forexample, we know that the cytosol contains many types of ions. Withwater having the ability to bond to a polar protein surface, you cancertainly expect a competition to arise between the water and the ionsfor the surface charge of the protein. HOWEVER, the concentration ofthe cells most abundant ion - potassium - is only 0.1 molar, whereasthe concentration of cellular water is on the order of 55 molar! With550 water molecules for every potassium ion, it is clear that the

presence of ions along the charged protein surface would be limited,although they certainly do exist.

We can also safely conclude that there would be more sodium andpotassium in an area of the cytosol where no proteins reside, and lessof these ions where the proteins do exist. However, some ions do getto aDsorb (not aBsorb) onto the protein surface. Now, just when youthink youve had enough folks, I must mention that sodium andpotassium are not the same size, in part, because of the size of theirrespective hydration shells. Thus, their respective charge densities aredifferent. What does this mean? This translates into more potassium

than sodium adsorbing onto the proteins. This in turn will create aconcentration gradient for various ions like potassium and sodiumwhich I also believe contributes to the differential exclusion of sodiumvs. potassium from the cell. (An additional; fact: sodium andpotassium each have six water molecules forming their respectivehydration shells. Yet because sodium is small than potassium, it takesmore hydration energy to remove the hydration shell around sodium,due to the positive influence of the closer nucleus of the ion. Thus, as


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stated above, this translates into more potassium than sodiumadsorbing onto proteins, again translating into more potassium thansodium adsorbing onto the surface of proteins and thus an ion gradientforming.)

Yes, I know you have learned that the sodium/potassium pump isresponsible for the concentration gradient that results in morepotassium inside the cell membrane and more sodium outside thatmembrane. BUT, remember that pump requires ATP; and it is myhumble opinion that most of the cells ATP production would have to beused for that purpose alone, were there not another mechanism ofdifferentially distributing ions on either side of the cell membrane - theion gradient created by surface charges on proteins and otherbiomolecules. Well, thats one mans (my) opinion, anyway. By theway, the staggering amount of surface area offer by cellular proteins isapproximately a staggering 80,000 square microns in a 16 micron cell!

Imagine that and its implications!

So we have bulk water, we have water organized to be ice-like, andwe have vicinal water along protein surfaces. When we get to thesubject of protein folding, we will see that water molecules engaging inhydrophobic interactions inside the inner, hydrophobicmicroenvironment of proteins will associate with themselves to formcages. These cages of water are called clathrates and will contributeto the energy and entropy changes that allow a protein to lose entropyand organize to fold. Thats for another lecture, however, so keepwhat I just explained to you at least on the back burner..

See! It didnt take long before I brought you back to the E word energy. Like all interactions in biochemistry, the essence ofinteractions between molecules and even within the same molecule isenergy. That is why I needed to introduce you to thermodynamicsearlier. Without those laws, we can not understand molecularstructure, enzyme catalysis, or bond formation. We will revisit thisagain when we discuss enzymes. How are we doing so far? You mightneed to read this a few times, but it should work for you. If it stilldoesnt, email me or pose your questions on the Discussion Board.

Before we go to our final topic regarding the aqueous aspects ofcellular biochemistry, I refer you to figure at the end of this lecturewhich I hope will pull the energy of things together for us. Thisreiterates, using a specific set of chemical equations, what I said aboutthe enthalpy change (H) in a chemical reaction contributing to theformation of dissipating heat and at the same time some useful energyG. The figure shows a respiration reaction vs. a full combustionreaction of the same fatty acid. Note that full combustion produces an


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exothermic reaction (-H = 2,340 kcal). That is why heat is written asa product in that reaction. However, in the respiration reaction, only1,384 kcal of heat are given off, because the remainder of the original2,340 kcal is converted into useful chemical energy (-G) in whichwork has been done that is, ATP is formed from ADP + phosphate.

This example should help clarify what we have been saying about therelationship of G to H; and it illustrates the concept that there is nosuch thing as a free lunch in biochemistry. Somebody payssomeplace. If entropy increases (+S) in one place, somewhere else itdecreases accordingly (-S). If energy is consumed at a given cellularlocation (+H), it is given off (-H) somewhere else. If a reaction isendergonic (+G) in one direction, it is exergonic (-G) in the oppositedirection and to the same degree or arithmetic value. OK?Having looked at the significant amount of dissipating heat that isproduced in just one set of chemical reactions, you can just now start

to imagine the total amount of heat produced by all of ourbiochemistry occurring simultaneously in each cell, let alone tissue,organ, and organism. Without exaggeration, the heat continuouslyproduced from our musculature alone would literally melt us anddenature just about every body protein. However, going back toelementary chemistry, the high specific heat of water (the amount ofheat required to raise 1 gram of water 1oC) allows our bodytemperature (the heat produced from all biochemical reactions) toremain relatively constant, because water can absorb a great deal ofheat without showing a significant temperature change.Needless to say, this makes a life-and-death difference.

Acids, Bases, and Buffers

Your pre-requisite to this course should have provided you with a goodbackground in these topics now to be mentioned. Therefore, I willtouch on them briefly and specifically where they are relevant to ourdeliberations in this course. So lets remember that an acid is just thatbecause it is a proton donor (H+) (a hydrogen atom which has lost itselectron) to another molecule. That would make water a theoreticalacid, although an extremely weak one. Let us agree that when we usethe term proton, we are ultimately speaking of the hydronium ion

(H3O+), because protons do not remain as such in aqueous solutions,including the colloidal cytosol of the cell, but rather bond to theelectronegative oxygen of water to form that hydronium ion. Whereaswe tend to think of a base as donating a hydroxyl ion (OH-), for thepurposes of this course, we are more interested in the Bronsted-Lowrydefinition of a base, that is, a substance that can accept an H+ from anacid. That would make water potentially a weak base, besides it beinga weak acid.


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The importance of this definition of a base is seen, for example, in thekidney, which helps regulate acid/base balance. Ammonia (NH

3) is

present in the kidney filtrate, and it can act as a base by combiningwith (removing) protons to form ammonium ion (NH

4+), which is

excreted in the urine. In its counterpart, a free amino acid or a fattyacid are acids, because they can donate a proton from their respectivecarboxyl groups (COOH) to form a carboxyl ion (COO-). Any free aminegroup of an amino acid can act as a base in the same capacity as justdescribed above for ammonia.

Although hydrochloric acid secreted from the parietal cells in thestomach is obviously a very strong acid, in that it can dissociate andgive up about 100% of its protons, most acids in the body are relativelyweak, dissociating but a small percentage of their protons.Most texts use the standard format for the dissociation constant of an

acid, where HA is the undissociated acid and A- represents theconjugate base (the acid minus its proton). Therefore, the equilibriumexpression will define the Ka (dissociation constant) of the acid by thefollowing formula:

Ka

= [H+] [A-]/[HA]

Note: the brackets [ ] designate concentration of

It should be intuitively obvious that the larger the Ka, the stronger the

acid (because a greater percentage of protons have been dissociatedfrom the acid) and the lower the pH, all other things being equal. Thisfits into the definition of pH as being the negative log of the [H+]. Inother words, pH = - log [H+]. I am sure you probably know this, so thisis just a reminder.

However, we need to understand this, in order to grasp thesignificance of the very important Henderson-Hasselbach equation.Page 9 of Devlin shows the derivation of this equation, or thisequation is easily found on line. However, for our purposes, youneed to understand the equation which defines the important pK

aterm,

where pH = pKa + log [A-

]/[HA].

Look at the log term. If the log [A-]/[HA] were = 1.0, then the termwould drop out, because the log of 1 is zero. For that to happen, youwould need the same amount of conjugate base [A-] as undissociatedacid [HA]. For that to happen, you would need 50% dissociation of theacid. If that happens, then the pK

a= pH, but only under those


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conditions. So now we have the important definition of pKa. It is the

pH at which there is 50% dissociation. You will see the real importanceof this when we look at amino acids, wherein we are interested indefining the pH at which a COOH group is 50% dissociated into COO-

and H+, and where an NH4

+ group is 50% dissociated into NH3

and H+.

pKa values can also, under certain circumstances, be applied to othergroups, such as the SH group in the amino acid cysteine as part of thestructure of the important antioxidant glutathione.

Buffers: We all know that buffers are present in cells and in the bloodfor the purpose of maintaining pH within a relatively narrow range.This is particularly necessary for the proper structure and function ofenzymes, and therefore metabolism. Remember, when we say pH, weare referring to the concentration of protons. In the human body,normal H+ levels lie between 35 45 nmol/L, with values < 20nmol/L

and > 120 nmol/L usually being incompatible with life. Clinically, thesevalues are sometimes reported as nmol/L values, rather than as pH.Normal body pH is, of course, about 7.34 - 7.43.

It is an interesting aside that over the course of a day, protonsresulting from normal oxidative metabolism (especially from digestedprotein rich in the sulfur-containing amino acids, cysteine andmethionine) are produced in millimolar amounts. This is about 105

times the normal blood concentration (present in nanomolar amounts).Therefore, the kidney is critical to maintaining acid/base balance byexcreting excess H+ in the manner I described above. Thus, forexample, one could then expect to observe a highly acidic urine from adiet high in animal protein.

Having diverted a bit from the buffer headline of this section, let usnow remind ourselves of the chemical definition of a buffer. It is aweak acid plus its conjugate base. In the human body, the mostprofoundly effective buffer is the bicarbonate buffer system. Otherweak acids and their respective conjugate base have some bufferingcapacity, but they are far less significant. Although I am going to defera complete explanation of the bicarbonate buffer system until wediscuss hemoglobin, I want to be sure we understand the how a bufferworks, whether in a test tube or in a living system.

Basically, a buffer is composed of a weak acid and its conjugate base(HA + A-). The weak acid, remember, means that there is littledissociation, and that the HA component of the buffer is mostly intact(undissociated). If the buffer system is of sufficient concentration andis not overloaded, in simple terms, this is how it works. Excess protons


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added to the system are neutralized by the A- component to form theHA form (little of which will dissociate). Should OH- (or another base)enter the system (as in a test tube titration), the HA will donate H+ toneutralize the OH- to form water.

Now if we do an old fashion titration curve, where we plot the amountof base added to a buffer against the change in pH, we will see thatwithin one pH unit of the pKa (+ or -), the buffer pH changes the least.(You can look up titration curves very easily on line.) Thus, we want abuffer that is approximately within that range of the pKa, in order for itto be most effective in its buffering capacity. That is one main reasonwhy our bodys main buffer system is the bicarbonate buffer, inaddition to its high concentration. By contrast, even though ammoniain the urine does accept protons to form ammonium ion, and thushelps control pH in the kidney, over all the ammonia/ammonium ionbuffer is not a good systemic buffer, because its pKa is about 9.3, and

our bodys pH is about 7.3. Well, for now, this is all we need toremember.

Closing Personal Note:

Part of what I have presented in this lecture might elicit the reactionfrom some of you, Oh, I have had a lot of this before. Others mightsay, however, I am never going to get through this course - to whichI respectfully reply, Oh yes you will. That is what I am here for. Soeither way, I have always felt that some review at the beginning of acourse is appropriate. Not everyone in the class has the same

background or same recent background, and I have never seen somereview to be counterproductive. However, that having been said, I amconfident that you shall be challenged over the course of thissemester. Again, remember that I feel very strongly about theimportance of the three concepts with which I began this lecture. Theyare a biochemical priority for me, because they begin to show the out-of-the box approach to this subject that is required for you to begin tomaster cellular metabolism. The details will quickly become part ofyour biochemical armamentarium; but dont lose the forest for thetrees, and respect the complexity of the living cell, should you betempted to erroneously search for simple summaries to complex

theory and even more complex clinical functions and solutions. Goodluck, and thanks for giving me the opportunity to share with you theunbelievably complex, multi-level, multi-dimensional, synergistic worldof biochemistry where the whole is truly greater than the sum of theparts.

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Introductory Concepts 560D on-line Updated