CHM333 LECTURES 6 & 7: 9/9 9/14 FALL 2009 Professor Christine Hrycyna Physiological Buffers All about maintaining equilibrium Major buffer in blood (ph 7.4) and other extracellular fluids is the carbonic acid/bicarbonate pair (See Window on Biochemistry 2 1 p.57 3rd edition) The ph of blood (7.4) is at the upper limit of the buffering capability of this system. Seems illsuited and inefficient But, we have a reserve supply of CO2 in our lungs which can replenish the H2CO3 Depends on three equilibria: Gaseous CO2 dissolves in blood to form carbonic acid Carbonic acid rapidly dissociates to H+ and bicarbonate o Concentration of carbonic acid in blood is very low Works in reverse as well, H+ is removed from cells by blood plasma o Neutralized by the reaction with HCO3 and leads to release of CO2 as gas from lungs Great system because it can be readily REGULATED! o CO2 regulated by breathing o HCO3 regulated by kidneys 30
MEDICAL CONDITIONS ASSOCIATED WITH BLOOD ph: Alkalosis Acidosis ALKALOSIS: Characterized by increase in blood ph (ph 7.74) Becomes more basic/alkaline CAUSES: Respiratory Alkalosis: o Hyperventilation: Breathing rate more rapid than necessary for normal CO 2 elimination Central nervous system disorders such as meningitis, encephalitis, cerebral hemorrhage Drug or hormone induced physiological changes Anxiety o Excessive intake of O 2 ; Abnormally low CO 2 due to excessive exhalation o ph goes so high that weakness and fainting result Hyperventilation causes [CO 2 ] Equilibrium shifts to the LEFT [H + ] goes down and ph goes UP TREATMENT: Correct underlying physiological problem In short term, respiratory alkalosis can be helped by breathing a CO 2 rich atomosphere (breathing into a paper bag) NH 4 Cl infusion (for alkalosis). NH 4 Cl dissociates into NH 4 + and Cl. The NH 4 + (ammonium) is in equilibrium with NH 3 (ammonia) and H +. Because ammonia is volatile, it is respired through the lungs, leaving behind H + and Cl 1 or hydrochloric acid, which lowers the ph. 31
ACIDOSIS: Blood ph goes DOWN [H + ] goes UP TWO TYPES: o Metabolic Acidosis Causes Uncontrolled diabetes Starvation diets Highprotein/low fat diets o Overproduce acidic compounds called ketone bodies that lower blood ph Sudden surges in LACTIC ACID during exercise o Respiratory Acidosis Causes Obstructive lung disease Hypoventilation (too much CO 2 ) Disease states that prevent efficient expiration of CO 2 [CO 2 ] (can t be expelled) Equilibrium shifts to the RIGHT [H + ] ; ph TREATMENT Treat the cause Stop alimentary loss of base; correct hypoxia; reduce renal acid load by diet; drain abscess in diabetic ketosis and give insulin; treat shock with intravenous fluids and stop hemorrhage etc In short term, can ventilate Commonly, bicarbonate is infused intravenousely (NaHCO 3 ) Other Physiological Buffering Systems: Hemoglobin: o Transports oxygen from lungs to peripheral tissues o Transports carbon dioxide from tissues to lungs for exhalation o Buffers blood by neutralizing H + and OH Phosphoric Acid Species o H 2 PO 4 and HPO 4 2 conjugate pair (pka = 7.2) (dihydrogen phosphate/monohydrogen phosphate) o Used a lot in labs to mimic cellular conditions o Use the HH equation to prepare these buffers Ionizable groups on amino acids in proteins assist in buffering (e.g histidine) Amino Acids as Acids, Bases and Buffers: 32
Amino acids are weak acids All have at least 2 titratable protons (shown below as fully protonated species) and therefore have 2 pka s o αcarboxyl (COOH) o αamino (NH 3 + ) Some amino acids have a third titratable proton in the R group and therefore a third pka o Showing all protonated: 33
pka Table for amino acids: * First column (pka 1 ) = COOH * Second column (pka 2 ) = NH 3 + * Third column (pka R ) = R group H + AMINO ACIDS AS WEAK ACIDS: Properties of amino acids in proteins and peptides are determined by the R group but also by the charges of the titratable group. Will ultimately affect protein structure. Important to know which groups on peptides and proteins will be protonated at a certain ph. How do we do this?? Example Alanine 1. Draw the fully protonated structure Q: Which protons come off when? A: Look at pka table for amino acids Alanine has 2 pkas: αcooh (pka = 2.3) comes off first (has lower pka) αnh 3 + (pka = 9.9) Others come off SEQUENTIALLY in ascending order of pka. 34
2. Write out structures for sequential deprotonation and place pka values over the equilibrium arrows. Alanine Fully protonated 1 st proton removed 2 nd proton removed Net charge = +1 Net charge = 0 Net charge = 1 So, from looking at the net charges, at different ph s, amino acids can have different charges! Very important for protein structure!! Remember that the pka = ph when ½ of an available amount of an ionizable group is ionized. Let s take a look at the titration curve for Alanine Looks very much like what we saw for acetic acid last time except that it has 2 midpoints (pka s) one for each proton αcooh and αnh 3 + Flat parts of curve are BUFFERING REGIONS o +/ 1 ph unit from pka At beginning, all protonated Need one equivalent of base for each proton At each HALF equivalent = pka o 50% protonated/50% deprotonated At end all deprotonated 35
For our purposes, to determine whether the proton is ON or OFF at a certain ph use the following RULES o ph = pka Equal amounts of protonated and deprotonated species exist For example: Alanine at different ph s (see pka table) if ph is LESS than the pka of a particular group, that group will be predominantly protonated if ph is GREATER than the pka of a particular ionizable group, that group will be predominantly deprotonated At ph 1.5: ph is less than the pka of both the αcooh and the αnh 3 +, therefore, both protons are ON At ph 7: ph is greater than the pka of the αcooh H + OFF ph is less than the pka of the αnh 3 + H + ON At ph 10.5 ph is greater than the pka of the αcooh H + OFF ph is greater than the pka of the αcooh H + OFF Apply same rules if there are 3 titratable protons: 1. Determine what the pka s of the titratable protons are by looking at the pka table 2. Draw the structures and the equilibria representing the complete deprotonation of the amino acid a. Start with fully protonated and then remove in order of pka values b. Put pka values above equilibrium 3. Determine at the ph of interest whether the proton is ON or OFF using the above rules 36
For example, Aspartate (D, Asp): Asp has 3 titratable protons 1. pka s for the three groups (look at Table3.2) 2. Draw the structures from fully protonated to fully deprotonated Note that all amino acids are at one point, electrically neutral at some ph value. This ph = isoelectric point (pi) How do you calculate pi? 1. Draw out the complete ionization of amino acid 2. Determine net charge on each ionized form 3. Find the structure that has no net charge 4. Take the average of the pka s that are around the structure with NO NET CHARGE pi = pka 1 + pka 2 2 5. Note do NOT just take the average of all pka s. What about Asp?? pkas: 2, 3.9, 10 (from Table 3.2) pi = (2+3.9)/2 = 2.95 Amino acids can be separated on the basis of their charges at a certain ph 37