Introductory Concepts For Biochemistry 560B Dr.

Charles Saladino In this course, we shall be covering material that is contained within many texts and other reference sources. However, as your professor, I believe in the strongest of terms that my role should not only be to present to you biochemical principles and specifics, but to show you how to learn this material through understanding, conceptualizing, extrapolating, making correlations within it, so that you will be able to relate it to clinical sequelae with these thought processes continuing throughout this course. In other words, we need to memorize, but we also need to appreciate cellular biochemistry by understanding what it is doing and what biomolecular strategy is being used in the process. In this regard, besides book material, you shall feel my own touch upon this course. This means I have had to prioritize material for which years’ worth of courses can be taken. Thus, in practical terms, I am forced 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 and basically miraculous interplay of cellular chemistry occurs. If you choose to take advantage of this approach to learning, you could really appreciate and perhaps even love a course which might otherwise intimidate some. Enjoy this experience, and hopefully, we will be exploring 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 to biochemistry. Even though I might not refer to these concepts during a given lecture, please keep them in mind as we explore various topics. When possible, try to apply them to whatever subject we discuss. The first concept is relatively simple. I refer to it as the structure/function relationship. That is, each cellular component is so structured as to function most efficiently. That seems quite obvious and is a simple concept when you think, for example, of a bone not functioning properly when it is broken. However, when you consider cell structure, then it becomes more detailed. If we look at a cell membrane (and we will later on in the course) composed, in part, of various phospholipids, that membrane will have dynamic fluidity properties that are dependent, again in part, upon the fatty acid composition of those phospholipids. Thus, if we alter the fatty acid composition of the membrane (such as occurs when a hyperlipidemic state alters the composition of platelet membranes – surprised?), then we can expect the membrane to have a change in its biophysical

properties, including a different fluidity within the membrane structure. So why is this important? With receptors traversing the membrane from the outside of the cell into the cytoplasm, one could expect alterations of at least the transmembrane portion of that receptor, with a subsequent change in receptor function and subsequent cellular transignaling. This could ultimately affect specific metabolic pathways within that cell. Before the course is completed, I will hopefully have time to elaborate on this, perhaps by talking about some of the research I have done in this area. However, in the mean time, I will ask you to keep this important structure/function concept in mind throughout the course. This particularly applies to what I hope is some of your knowledge about topics such as the function-based three dimensional structure of proteins (especially enzymes), as well as to the electron transport system and the proton gradient that is generated along the inner mitochondrial membrane during oxidative phosphorylation (ATP production). Let me give a specific, heads-up examples regarding this topic: The first example refers to proteins that are embedded into the inner mitochondrial membrane at a precise distance apart that allows for the efficient transfer of electrons. This electron transport, as we shall see in a later lecture, generates the energy needed to drive protons across the space between the inner and outer mitochondrial membrane space. The protomotive force ultimately generates the energy required to form ATP. If a drug is used to expand the entire mitochondrion, then the inner membrane is stretched, the proteins are not at the proper distance for electron transfer, and ultimately ATP can not be generated. The other example I wish to use illustrates the structure/function concept at the most fundamental level known in the universe - that is, the quantum/electron level. So, during digestion, the enzyme lactase splits the sugar lactose into its components - glucose and galactose. In order for this to be accomplished, the lactase must fire a proton into the heart of the lactose. Now this particular proton is usually bonded to a key, obviously electronegative, oxygen. But sometimes, the proton 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 that the proton is on the oxygen and not the nitrogen????? Is this not incredibly amazing!!!

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

relationships than does the first principle with which we began our discussion. Let us refer to this second concept as compartmentaliziation. At the cellular level, we might define this as a localization of certain biochemical sequences which are often physically separated from other biomolecular reactions by highly hydrophobic membranes that help form specific structural barriers within various portions of the cell. Just stop for a moment, and consider this concept carefully. What does it really mean, and, therefore, what does it really imply? The answer is “a great deal.” Let us start here with some illustrative examples. We all remember from our most elementary courses that the mitochondria is where the great majority of the ATP is produced in cells that in fact contain those structures. Thus, a specific electrochemical environment is required for this ATP production, one that is quite different from that which is found in most other parts of the cell. Such an environment would not be particularly conducive to other chemical reactions that must take place in the free cytoplasm, for example. The electrochemical, oxidative/reductive environment is closely related to local pH, which is a critical factor in the way a protein folds, as well as in providing the proper environment to support a myriad of oxidation vs. reduction reactions. Thus, compartmentalization, therefore, allows for specific intracellular microenvironments that enhance the efficiency of specific biochemical pathways upon which a viable cell must depend. Another important point by way of example: There are numerous regulatory steps and specific molecules which govern the formation of ATP in the mitochondria. One of many is the availability of phosphate ion, as one would expect. The phosphate ion must cross into the mitochondria to be available for oxidative phosphorylation. By compartmentalizing the bulk of ATP formation to the mitochondria, easier and more efficient regulation of that process can occur by (in part) controlling access of critical components (like phosphate ion) to the inner mitochondrial membrane, which is where ADP is converted into ATP. Further, responding to the need for this high energy compound in other parts of the cell, egress of ATP from the mitochondria can be regulated more easily. One more example: The cellular lysosome is a single-membrane bound structure which contains a large spectrum of hydrolytic enzymes used to break down various biomolecules, whose components are likely to be recycled etc. If those enzymes were allowed to be activated without being compartmentalized, severe cellular damage could result in a random manner, as in fact does occur under aberrant conditions. This is but one of many examples of isolating reactions that

can not be mixed into an environment with other reactions. Before closing this topic, I would like to ask you a few rhetorical questions very much related to compartmentalization. If I were to ask you why we aren’t one big ameba, you might laugh and say you actually think you know someone who is! However, seriously, what I am really asking you is why we aren’t one giant cell? Why are we so terribly multicellular? I am sure many of you would say, “Oh, cell specialization,” or perhaps, “It is more efficient.” Those would be true statements, but do they really get at the heart of compartmentalizing various bodily functions to specific cells and thus tissues and organs? Not really. After all, one could argue that an ameba or paramecium has some type of neural-like response, feeds, digests, has sex, etc. Of course I am certainly not implying that we are no better off than a single cell organism, especially with our wonderful brain and the fantastic 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 from the outside of your nose to where your heart would be, if we were one big cell. Ask yourself how long it would take to transignal a metabolic stimulus from a receptor on the surface of your skin to a metabolic reaction located somewhere in the middle of you body? The answers now become obvious. It could never happen that way. Why? Contemplate this. This should really get you thinking. Might you consider surface area to volume ratios? Think about how much greater a surface area a hummingbird has than an elephant when compared to their respective volumes. That ratio is much greater in a hummingbird, and I ask you which has the higher metabolic rate? Who has a greater metabolic rate and has less problem keeping off weight (all other things being equal), a 6’5” 250 lb person, or one who is 4’5” and 250 lb? Obviously, the answer is the person with a high surface area to volume ratio. Make sense? Thus, in the mean time and in summary, compartmentalization provides for greater cellular control of its own biochemistry than would otherwise be the case, and it increases the surface area to volume ratio to provide more surface area upon and in which reactions can occur. Of great importance is the fact that this translates into greater efficiency with which cellular function can take place. At the heart of the third concept to which I wish to introduce you is this extremely important term “efficiency.” Unless otherwise stated, you can correctly assume the word “efficiency” to mean energy being utilized 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

energy as possible, understanding that no machine - physical or chemical - is 100% efficient. The use and production of cellular energy is governed by the same laws 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 is the Second Law of Thermodynamics, with which I hope you have some familiarity. We absolutely must appreciate the relationship of its terms to each other, if we are understand why we really need to consume particular nutrients, how energy-requiring metabolic reactions are able to occur, and what happens to some of the energy derived from those reactions that produce it. This inter-relationship is absolutely critical to maintaining a viable living system. So let’s start. ΔG = ΔH – TΔS As I am sure you already know, the Δ symbol refers to a “change in” for all terms to which it is attached. Thus: ΔG = free energy change – the energy available to do work ΔH = enthalpy or heat content change T = temperature in degrees Kelvin ΔS = entropy (disorder, randomness, chaos) change If we look at the overall equation and try to summarize it, we should conclude that the natural tendency of the universe is to move toward randomness or disorder (a more + ΔS term). That applies to macromolecules, cellular structure, and the whole organism. How fast entropy (disorder, chaos, randomness) is approached and ultimately achieved can be directly modulated by the temperature and how much free energy is available. We can dispense with the T in this course, because we can not substantially alter our body temperature (in degrees Kelvin) sufficiently enough affect a change in entropy, as we certainly could do in a test tube reaction to which heat is easily added. Simply stated, we are warm-blooded organisms. I will not spend much time on the ΔH term either, which represents the difference in heat content of reactants vs. products of a reaction. However, please remember that an endothermic reaction (where ΔH is positive) requires energy to make that reaction go. On the other hand, an exothermic reaction (where ΔH is negative) produces heat. Of that heat produced, some is dissipated and is absorbed by the water contained in the cell, contributing to our constant body temperature (because water has a high specific heat). The rest of that exothermic energy can be utilized to do work. This useful energy (that which is

available to do work) is known as the free energy (the G term) of a reaction and is a form of energy upon which countless, life-sustaining biochemical reactions rely, in order to go to completion. It is now very important to our understanding of metabolism that we carefully look at this ΔG term. With the appreciation that the value of ΔG is dependent upon all the other terms in the Second Law Equation shown above, we must know whether that ΔG term is positive or negative. This is absolutely critical to understanding metabolism and is not just a theoretical exercise that requires arithmetic. In brief, if the ΔG term is positive, then the reaction is said to endergonic and will not occur spontaneously. In significant contradistinction, a reaction is said to be exergonic, and thus spontaneous, when the ΔG value is negative. In chemistry we define spontaneous a little differently than one might use it in straight vocabulary. Spontaneous means that a reaction 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 of energy input, in order to complete the reaction. We will investigate this in much more detail when we discuss enzyme kinetics, as well as energy biotransformation. For now, however, let us clearly delineate the idea that anabolic reactions (where biomolecules are synthesized) are most likely to be endergonic, whereas catabolic reactions (where biomolecules are broken down into simpler components) are probably going 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 Kreb’s cycle (which we certainly will cover in this course), as it passes through each of its steps, is never reversible as a whole cycle. The answer is that the direction the Kreb’s cycle takes is because the cycle as a whole has a large negative ΔG. In fact, almost every step has a negative ΔG value. Reversing the Kreb’s cycle would not be spontaneous, and thus would require energy (and the enzymes) that would simply not be available. This is no accident. We can close this major (third) concept of the Second Law by pointing out that ultimately, entropy wins out, and individual cells, and finally the whole organism, dies. This represents disorder, compared to vibrant living systems, which display more order (less randomness or less disorder). The higher the negative ΔS value, the greater chance order or structure will need to be maintained by an imput of free energy from somewhere. So the chaos or disorder term relies upon the value of the ΔG term. In other words, this does not occur in a vacuum. 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

work of synthesis and the maintenance of cell structure and function. Take the extreme case of starvation. Little by little, without a renewed energy input (negative ΔG), the cells and the organism approach greater and greater positive ΔS (randomness, disorder, chaos), and the organism 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 spontaneously and without looking at any reference material. Now I am certainly not saying this so as to pat myself on the back – hardly. Rather, my point is that I consider the above material so fundamental to understanding many of the “whys” of biochemistry, that I carry these thoughts around with me whenever I consider human metabolism and nutrition. Therefore, I am saying to you, make sure you fully grasp what I have just written here. Keep it with you; and attempt to apply these principles as we discuss the biochemistry of human nutrition. Last, but not least: Please don’t allow yourself to fall into the quicksand of thinking that, “I will never use this in my practice.” If you do, then you will miss the opportunity to critically analyze a great deal of important future data presented in the scientific literature. Believe me, if you grasp these underlying principles of metabolism, you will be a better health care provider, because you will think critically at a higher level. In other words, I would simply offer to you the thought that one should strive not to be a technician only in their practice, and that the empirical approach to reading the biomedical literature and to treating a patient is not the superior one, when compared to having the understanding of and appreciation for the depth of the cellular and molecular inter-relationships that are biochemistry. So let’s continue…….. The Aqueous Environment We all know that the living cell contains a very large percentage of water by weight, depending upon the cell, of course; and this is also the case in the various extracellular compartments. Although a discussion of the details of the many roles of water in terms of fluid balance, osmotic considerations, etc. is not appropriate for this course, we do need to consider the aqueous compartment of cells because of the fact that water is a polar molecule (with oxygen being more electrophilic than hydrogen, finalizing a relatively positive change at the hydrogen atoms and a relatively negative charge at the oxygen). Obviously then water is very cohesive, because it hydrogen bonds with other water molecules. It would then follow that such cohesiveness would dramatically affect interactions between molecules in solution. Specifically, the polarity and hydrogen bonding capability of water

make it highly interactive, in that water is an excellent solvent for polar molecules. The reason for this is that water weakens the electrostatic and hydrogen bonding between polar molecules (including ions) by competing for their attractions, which is why non-polar molecules are water-insoluble. What does this mean in practical terms? I would think the existence of life depends critically on the capacity of water to dissolve a remarkable array of molecules that include fuels, building blocks, catalysts, and carriers of information. Point: This means that high concentrations of these polar molecules can coexist at the same time in the water environment, where they can diffuse around and interact with one another. You might want to ask this question, if you are doing some real thinking. Because water would appear then to weaken interactions between polar molecules, wouldn’t this cause a problem? Well, another of what will be many examples of a truly incredible design is that many proteins can create a water-free environment within a protein (yes a microenvironment within a molecule – contemplate that), which would allow for hydrophobic interactions within the protein to occur. Thus, the posed problem is circumvented. We will see more of this in detail when we study protein structure. We will also see later on that water actually helps drive the folding of proteins by helping with 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 you need. However, I am presenting the following to start to develop more contemplative thinking skills, and to give you a better appreciation of how very, very complex the topic of even the simplest ions can be when it comes to the aqueous environment. So here goes: Now when we say the aqueous environment, we have a tendency to automatically think of water as it would come out of kitchen faucet. To be sure, some of the cellular water does indeed take this form with an orderly hydrogen bonding mediating the organization of what we will call “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 under certain conditions is found to be more hydrogen bonded than bulk water, although not as much as what we would observe in ice. This should begin to spark your thinking, because if our very polar water is organized (hydrogen bonded to itself) to different degrees in different parts of the cytoplasm (which is the case), then it would

follow that the various forms of water create certain microenvironments. It would also follow that ions like sodium and potassium would not be distributed homogeneously throughout the cell cytosol and, thus, neither would the electrochemical environment. Let me explain that a bit. Think about a cellular protein with its surface charges arising from the various amino acid carbonyl (-) and amino (+) groups and the R groups (side chains) (+ or -). Further, you would expect each amino acid comprising the surface of the protein to present several charges. These charges would have a substantial effect upon water, which being a 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 along the protein’s charged surface - say the positive side of water to the negative charge on the protein (or vise versa). In addition, because of the dipolar nature of water, we can expect layers of water to form along at least some of the surface of proteins, with the layering limited by the distance of the water from the charged protein surface. The implications of all this are quite staggering. Think about it. For example, we know that the cytosol contains many types of ions. With water having the ability to bond to a polar protein surface, you can certainly expect a competition to arise between the water and the ions for the surface charge of the protein. HOWEVER, the concentration of the cell’s most abundant ion - potassium - is only 0.1 molar, whereas the concentration of cellular water is on the order of 55 molar! With 550 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 and potassium in an area of the cytosol where no proteins reside, and less of these ions where the proteins do exist. However, some ions do get to aDsorb (not aBsorb) onto the protein surface. Now, just when you think you’ve had enough folks, I must mention that sodium and potassium are not the same size, in part, because of the size of their respective hydration shells. Thus, their respective charge densities are different. What does this mean? This translates into more potassium than sodium adsorbing onto the proteins. This in turn will create a concentration gradient for various ions like potassium and sodium which I also believe contributes to the differential exclusion of sodium vs. potassium from the cell. (An additional; fact: sodium and potassium each have six water molecules forming their respective hydration shells. Yet because sodium is small than potassium, it takes more hydration energy to remove the hydration shell around sodium, due to the positive influence of the closer nucleus of the ion. Thus, as

stated above, this translates into more potassium than sodium adsorbing onto proteins, again translating into more potassium than sodium adsorbing onto the surface of proteins and thus an ion gradient forming.) Yes, I know you have learned that the sodium/potassium pump is responsible for the concentration gradient that results in more potassium inside the cell membrane and more sodium outside that membrane. BUT, remember that pump requires ATP; and it is my humble opinion that most of the cell’s ATP production would have to be used for that purpose alone, were there not another mechanism of differentially distributing ions on either side of the cell membrane - the ion gradient created by surface charges on proteins and other biomolecules. Well, that’s one man’s (my) opinion, anyway. By the way, the staggering amount of surface area offer by cellular proteins is approximately 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,” and we have vicinal water along protein surfaces. When we get to the subject of protein folding, we will see that water molecules engaging in hydrophobic interactions inside the inner, hydrophobic microenvironment of proteins will associate with themselves to form cages. These cages of water are called clathrates and will contribute to the energy and entropy changes that allow a protein to lose entropy and organize to fold. That’s for another lecture, however, so keep what I just explained to you at least on the back burner.. See! It didn’t take long before I brought you back to the E word – energy. Like all interactions in biochemistry, the essence of interactions between molecules and even within the same molecule is energy. That is why I needed to introduce you to thermodynamics earlier. Without those laws, we can not understand molecular structure, enzyme catalysis, or bond formation. We will revisit this again when we discuss enzymes. How are we doing so far? You might need to read this a few times, but it should work for you. If it still doesn’t, email me or pose your questions on the Discussion Board. Before we go to our final topic regarding the aqueous aspects of cellular biochemistry, I refer you to figure at the end of this lecture which I hope will pull the “energy of things” together for us. This reiterates, using a specific set of chemical equations, what I said about the enthalpy change (ΔH) in a chemical reaction contributing to the formation of dissipating heat and at the same time some useful energy ΔG. The figure shows a respiration reaction vs. a full combustion reaction of the same fatty acid. Note that full combustion produces an

exothermic reaction (-ΔH = 2,340 kcal). That is why heat is written as a product in that reaction. However, in the respiration reaction, only 1,384 kcal of heat are given off, because the remainder of the original 2,340 kcal is converted into useful chemical energy (-ΔG) in which work has been done – that is, ATP is formed from ADP + phosphate. This example should help clarify what we have been saying about the relationship of ΔG to ΔH; and it illustrates the concept that there is no such thing as a “free lunch” in biochemistry. Somebody pays someplace. If entropy increases (+ΔS) in one place, somewhere else it decreases accordingly (-ΔS). If energy is consumed at a given cellular location (+ΔH), it is given off (-ΔH) somewhere else. If a reaction is endergonic (+ΔG) in one direction, it is exergonic (-ΔG) in the opposite direction and to the same degree or arithmetic value. OK? Having looked at the significant amount of dissipating heat that is produced in just one set of chemical reactions, you can just now start to imagine the total amount of heat produced by all of our biochemistry occurring simultaneously in each cell, let alone tissue, organ, and organism. Without exaggeration, the heat continuously produced from our musculature alone would literally melt us and denature just about every body protein. However, going back to elementary chemistry, the high specific heat of water (the amount of heat required to raise 1 gram of water 1oC) allows our body temperature (the heat produced from all biochemical reactions) to remain relatively constant, because water can absorb a great deal of heat 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 good background in these topics now to be mentioned. Therefore, I will touch on them briefly and specifically where they are relevant to our deliberations in this course. So let’s remember that an acid is just that because it is a proton donor (H+) (a hydrogen atom which has lost its electron) to another molecule. That would make water a theoretical acid, although an extremely weak one. Let us agree that when we use the 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 the electronegative oxygen of water to form that hydronium ion. Whereas we tend to think of a base as donating a hydroxyl ion (OH-), for the purposes of this course, we are more interested in the Bronsted-Lowry definition of a base, that is, a substance that can accept an H+ from an acid. That would make water potentially a weak base, besides it being a weak acid.

The importance of this definition of a base is seen, for example, in the kidney, which helps regulate acid/base balance. Ammonia (NH3) is present in the kidney filtrate, and it can act as a base by combining with (removing) protons to form ammonium ion (NH4+), which is excreted in the urine. In its counterpart, a free amino acid or a fatty acid are acids, because they can donate a proton from their respective carboxyl groups (COOH) to form a carboxyl ion (COO-). Any free amine group of an amino acid can act as a base in the same capacity as just described above for ammonia. Although hydrochloric acid secreted from the parietal cells in the stomach is obviously a very strong acid, in that it can dissociate and give up about 100% of its protons, most acids in the body are relatively weak, 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 the conjugate base (the acid minus its proton). Therefore, the equilibrium expression will define the Ka (dissociation constant) of the acid by the following 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 dissociated from the acid) and the lower the pH, all other things being equal. This fits into the definition of pH as being the negative log of the [H+]. In other words, pH = - log [H+]. I am sure you probably know this, so this is just a reminder. However, we need to understand this, in order to grasp the significance of the very important Henderson-Hasselbach equation. Page 9 of Devlin shows the derivation of this equation, or this equation is easily found on line. However, for our purposes, you need to understand the equation which defines the important pKa term, where pH = pKa + log [A-]/[HA]. Look at the log term. If the log [A-]/[HA] were = 1.0, then the term would drop out, because the log of 1 is zero. For that to happen, you would need the same amount of conjugate base [A-] as undissociated acid [HA]. For that to happen, you would need 50% dissociation of the acid. If that happens, then the pKa = pH, but only under those

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 importance of this when we look at amino acids, wherein we are interested in defining the pH at which a COOH group is 50% dissociated into COOand H+, and where an NH4+ group is 50% dissociated into NH3 and H+. pKa values can also, under certain circumstances, be applied to other groups, such as the SH group in the amino acid cysteine as part of the structure of the important antioxidant glutathione. Buffers: We all know that buffers are present in cells and in the blood for the purpose of maintaining pH within a relatively narrow range. This is particularly necessary for the proper structure and function of enzymes, and therefore metabolism. Remember, when we say pH, we are 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, these values 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, protons resulting from normal oxidative metabolism (especially from digested protein rich in the sulfur-containing amino acids, cysteine and methionine) 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 by excreting excess H+ in the manner I described above. Thus, for example, one could then expect to observe a highly acidic urine from a diet high in animal protein. Having diverted a bit from the buffer headline of this section, let us now remind ourselves of the chemical definition of a buffer. It is a weak acid plus its conjugate base. In the human body, the most profoundly effective buffer is the bicarbonate buffer system. Other weak acids and their respective conjugate base have some buffering capacity, but they are far less significant. Although I am going to defer a complete explanation of the bicarbonate buffer system until we discuss hemoglobin, I want to be sure we understand the how a buffer works, 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 little dissociation, and that the HA component of the buffer is mostly intact (undissociated). If the buffer system is of sufficient concentration and is not overloaded, in simple terms, this is how it works. Excess protons

added to the system are neutralized by the A- component to form the HA 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+ to neutralize the OH- to form water. Now if we do an old fashion titration curve, where we plot the amount of base added to a buffer against the change in pH, we will see that within 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 a buffer that is approximately within that range of the pKa, in order for it to be most effective in its buffering capacity. That is one main reason why our body’s main buffer system is the bicarbonate buffer, in addition to its high concentration. By contrast, even though ammonia in the urine does accept protons to form ammonium ion, and thus helps control pH in the kidney, over all the ammonia/ammonium ion buffer is not a good systemic buffer, because its pKa is about 9.3, and our body’s pH is about 7.3. Well, for now, this is all we need to remember. Closing Personal Note: Part of what I have presented in this lecture might elicit the reaction from some of you, “Oh, I have had a lot of this before.” Others might say, however, “I am never going to get through this course” - to which I respectfully reply, “Oh yes you will. That is what I am here for.” So either way, I have always felt that some review at the beginning of a course is appropriate. Not everyone in the class has the same background or same recent background, and I have never seen some review to be counterproductive. However, that having been said, I am confident that you shall be challenged over the course of this semester. Again, remember that I feel very strongly about the importance of the three concepts with which I began this lecture. They are a biochemical priority for me, because they begin to show the outof-the box approach to this subject that is required for you to begin to master cellular metabolism. The details will quickly become part of your biochemical armamentarium; but don’t lose the forest for the trees, and respect the complexity of the living cell, should you be tempted to erroneously search for simple summaries to complex theory and even more complex clinical functions and solutions. Good luck, and thanks for giving me the opportunity to share with you the unbelievably complex, multi-level, multi-dimensional, synergistic world of biochemistry where the whole is truly greater than the sum of the parts.

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