BIOCHEM REVIEW #1

1. BIOCHEM REVIEW #1 Jason Emer jemer1@uic.edu Obi Ekwenna oekwen1@uic.edu

2. YOUR TEST • Monday, September 13 • ~2-3 questions per Lec. (26-39) • ~Pass Level 55-65%

3. Acid-Base Chemistry • Acid: a proton donor • Base: accepts protons • HA <-----> A- + H+ • Ka = [H+][A-]/[HA] • pKa = -logKa

4. Acid-Base Chemistry • Weak acids and bases in solution do not fully dissociate and, therefore, there is an equilibrium between the acid and its conjugate base. • This equilibrium can be calculated and is termed the equilibrium constant = Ka. This is also sometimes referred to as the dissociation constant as it pertains to the dissociation of protons from acids and bases.

5. Acid-Base Chemistry • Why is this important? • pKa = pH -log[A-]/[HA] • By rearranging the above equation we arrive at the Henderson-Hasselbalch equation: pH = pKa + log[A-]/[HA]

6. Acid-Base Chemistry • Clinical Significance…….Blood Buffering • The pH of blood is maintained in a narrow range around 7.4. • Even relatively small changes in this value of blood pH can lead to severe metabolic consequences. • Therefore, blood buffering is extremely important in order to maintain homeostasis:Aka Ying Yang…..not the Twins.

7. Acid-Base Chemistry • The primary buffers in blood are hemoglobin in erythrocytes and bicarbonateion (HCO3-) in the plasma. • The formation of bicarbonate ion in blood from CO2 and H2O allows the transfer of relatively insoluble CO2 from the tissues to the lungs, where it is expelled. The major source of CO2 in the tissues comes from the oxidation of ingested carbon compounds

8. Acid-Base Chemistry

9. Acid-Base Chemistry • CO2 + H2O <------> H2CO3 • H2CO3 <-------> H+ + HCO3-

10. Acid-Base Chemistry • If blood is not adequately buffered, the result may be metabolic acidosis or metabolic alkalosis. • These physiological states can be reached if a metabolic defect results in the inappropriate accumulation or loss of acidic or basic compounds. • These compounds may be ingested, or they may accumulate as metabolic by-products such as acetoacetic acid and lactic acid. Both of these will ionize, thereby increasing the level of H+ ions that will in turn remove bicarbonate ions from the blood and alter blood pH. The predominant defect in acid or base elimination arises when the excretory system of the kidneys is impaired. • Alternatively, if the lungs fail to expel accumulated CO2 adequately and CO2 accumulates in the body, the result will be respiratory acidosis. If a decrease in PCO2 within the lungs occurs, as during hyperventilation, the result will be respiratory alkalosis.

11. Acid-Base Chemistry • Typical Questions: • What is the pKa of an acid which has a pH of 6.8 when its base:acid ratio is 1:20? Dang!!! We ‘ve to know that pH eqn! YES! • A patient comes into the emergency room feeling faint. He says that he is an insulin-dependent diabetic. His blood gases have been determined and you see his value for [HCO3-] = 18 miliequivalents per liter and for PCO2, = 38 mmHg. Based on your calculations this patient has? Acidosis? Alkalosis? Danger or Not?

12. Amino Acids, Polypeptides & Proteins • All peptides and polypeptides are polymers of alpha-amino acids. • There are 20 -amino acids that are relevant to the make-up of mammalian proteins. • Several other amino acids are found in the body free or in combined states (i.e. not associated with peptides or proteins).

13. Amino Acids, Polypeptides & Proteins • The -amino acids in peptides and proteins (excluding proline) consist of a carboxylic acid (-COOH) and an amino (-NH2) functional group attached to the - carbon atom.

14. Amino Acids, Polypeptides & Proteins • Each of the 20  -amino acids found in proteins can be distinguished by the R-group substitution on the alpha-carbon atom. • Two broad classes of amino acids based upon whether the R-group is hydrophobic or hydrophilic. • The hydrophobic amino acids reside predominantly in the interior of proteins. This class of amino acids does not ionize nor participate in the formation of H-bonds. • The hydrophilicamino acids tend to interact with the aqueous environment, are often involved in the formation of H-bonds and are predominantly found on the exterior surfaces proteins or in the reactive centers of enzymes.

15. Amino Acids, Polypeptides & Proteins • Grouping of Amino-Acids • Aliphatic Chain Amino Acids • Gly, Ala, Val, Leu, Ile • Non-Aromatic Amino Acids with -OH • Ser, Thr • Amino Acids with Sulfur-Containing R-Groups • Cys, Met • Acidic Amino Acids and their Amides • Asp, Asn, Glu, Gln • Basic Amino Acids • Arg, Lys, His • Amino Acids with Aromatic Rings • Phe, Trp, Try, Pro

16. Amino Acids, Polypeptides & Proteins • Acid-Base Properties of the Amino Acids • Key Points: • The -COOH and -NH2 groups in amino acids are capable of ionizing (as are the acidic and basic R-groups of the amino acids). • At physiological pH (around 7.4) the carboxyl group will be unprotonated and the amino group will be protonated. • An amino acid with no ionizable R-group would be electrically neutral at this pH. This species is termed a zwitterion. • As a general rule the amino terminal has a pKa~9.4 and carboxy-terminal is at pKa~2.0

17. Amino Acids, Polypeptides & Proteins • The net charge (the algebraic sum of all the charged groups present) of any amino acid, peptide or protein, will depend upon the pH of the surrounding aqueous environment. • As the pH of a solution of an amino acid or protein changes so too does the net charge. This phenomenon can be observed during the titration of any amino acid or protein. • When the net charge of an amino acid or protein is zero the pH will be equivalent to the isoelectric point: pI. • pI=(pKa1+pKa2)/2

18. Amino Acids, Polypeptides & Proteins which amino acid is this?

19. Amino Acids, Polypeptides & Proteins • The Peptide Bond • Amino acids can be joined together to form a peptide or polypeptide. They are called peptides because when the carboxyl group of one amino acid joins to the amino group of another, a peptide bond is formed. Chemically this is an amide bond but when it occurs in proteins it is given the name peptide bond. The partial double bond nature of the peptide bond means that there is not free rotation about the C -- N bond. The most stable conformation is planar and trans

20. Amino Acids, Polypeptides & Proteins • Peptide chain (a.k.a. polypeptide) has direction. • N-Asparagine-Glutamate-Glycine-C • What is the pI of this tripeptide? Be sure to know the three letter code of all the amino acids! • There is no set length of a polypeptide (how long is a piece of string) although most polypeptides in nature are between 50 and 2000 residues long. • So how do we determine the sequence of a polypeptide?

21. Amino Acids, Polypeptides & Proteins • Prior to sequencing peptides it is necessary to eliminate disulfide bonds within peptides and between peptides using 2-mercaptoethanol. • To determine N-terminus • Sanger Agent: 2,4-dinitrofluorobenzene (DNF) detected by yellow pigment observed via SDS-PAGE • Dansyl Chloride: Like Sanger however detected via Fluorescence. • Edman Degradation: sequential removal of amino terminal amino acid using phenylisothiocyanate. Now automated.

22. Amino Acids, Polypeptides & Proteins • Due to the limitations of the Edman degradation technique, peptides longer than around 50 residues can not be sequenced completely! So we have more stuff for you to remember! • Trypsin: cuts carboxyl terminal of LYS, ARG except Pro • Chymotrpsin: cuts carboxyl terminal of Aromatic, except Pro • Carboxypeptidase A: not specific; cuts carboxyl terminal of almost aas, except Lys & Arg, or if Pro is terminal residue • Carboxypeptidase B: not specific cuts carboxyl terminal of Lys & Arg

23. Amino Acids, Polypeptides & Proteins • Other techniques to keep in mind: • Cyanogen bromide (CNBr):This reagent causes specific cleavage at the C-terminal side of Met residues. The number of peptide fragments that result from CNBr cleavage is equivalent to one more than the number of Met residues in a protein. • The most reliable chemical technique for C-terminal residue identification is hydrazinolysis. A peptide is treated with hydrazine, NH2-NH2, at high temperature (90oC) for an extended length of time (20-100hr). This treatment cleaves all of the peptide bonds yielding amino-acyl hydrazides of all the amino acids excluding the C-terminal residue which can be identified chromatographically compared to amino acid standards. • Sample question: Answer True/False • Cynanogen Bromide will cleave this polypeptide into a 6-peptide chain and a tripeptide: • Glu-Ser-Thr-Phe-Met-Asn-Trp-Met

24. Proteins Protein Primary Structure • The primary structure of peptides and proteins refers to the linear number and order of the amino acids present. Has all the info to FOLD! • We can use the methods described earlier to determine the sequence. Also see Lecture 4 PowerPoint for other key factoids and concepts!

25. Proteins • Protein Secondary Structure • Local Confirmation • 1. Alpha-helix: The formation of the a-helix is spontaneous and is stabilized by H-bonding between amide nitrogen's and carbonyl carbons of peptide bonds spaced four residues apart. Disrupted by Proline • 2. Beta sheet: b-sheets are composed of 2 or more different regions of stretches of at least 5-10 amino acids. The folding and alignment of stretches of the polypeptide backbone aside one another to form b-sheets is stabilized by H-bonding between amide nitrogens and carbonyl carbons beta-Sheets are either parallel or antiparallel. • In parallel sheets adjacent peptide chains proceed in the same direction (i.e. the direction of N-terminal to C-terminal ends is the same), whereas, in antiparallel sheets adjacent chains are aligned in opposite directions. • There are also Super-secondary Structures (helix-turn-helix, helix-loop-helix and zinc finger domains of eukaryotic transcription factors); Do not worry about this much. For the gunners, Read Voet & Voet.

26. Proteins Tertiary Structure • See me in 3-D Baby! • Interaction between amino acid residues • Hydrophobic amino acids inside • Hydrophilic amino acids on the surface • 3-D maintained by hydrophobic, electrostatic & hydrogen interactions (non-covalent). • Also present could be disulfide bonds. Secondary structures of proteins often constitute distinct domains. Therefore, tertiary structure also describes the relationship of different domains to one another within a protein. See the structure of Ig!

27. Proteins Quaternary Structure • Many proteins contain 2 or more different polypeptide chains that are held in association by the same non-covalent forces that stabilize the tertiary structures of proteins. • The intrachain disulfidebond is the one covalent bond involved in maintenance of tertiary structure. • The interchain disulfide bond can be used to stabilize quaternary structure.

28. Carbohydrates • Carbohydrates are carbon compounds that contain large quantities of hydroxyl groups • carbohydrates that are of physiological significance exist in the D-conformation • Structures you must know! All of them! • But if nothing else know these: • Monosaccharide • Disaccharides • Complex Sugars

29. Carbohydrates • More Structures to Know: L-Fucose is rare L sugar found of the oligosaccharide chains of N- and O-linked glycoproteins.

30. Carbohydrates Maltose: the major degradation product of starch, is composed of 2 glucose monomers in an a-(1,4) glycosidic bond Lactose: is found exclusively in the milk of mammals andconsists of galactose and glucose in a b-(1,4) glycosidic bond

31. Carbohydrates Sucrose: prevalent in sugar cane and sugar beets, is composed of glucose and fructose through an a-(1,2)-glycosidic bond Polysaccharides Carbohydrates found in nature occur in the form of high molecular weight polymers called polysaccharides. Ex: Glycogen; alpha 1,4 linkage and alpha 1,6 branching of glucose (animals) Starch: amylose only alpha 1,4 linkage of glucose not as compact as glycogen Amylopectin: has branching alpha 1, 6 in addition to 1,4 linkage. Be aware of the uses of CHOs in biological systems, Memorize the glucosaminoglycans pointed out in lecture, know the structures of O, A, B blood antigens, and how to distinguish each structure. O is universal donor, AB universal acceptor, etc.

32. Nucleic Acids Nucleotides may be considered one of the most important metabolites of the cell. Nucleotides are found primarily as the monomeric units comprising the major nucleic acids of the cell, RNA and DNA. Used as energy stores ATP, NADH, NADPH, NAD+, NADP+, FAD, FADH2, etc cAMP and other second messengers

33. Nucleic Acids • The nucleotides found in cells are derivatives of the heterocyclic highly basic, compounds, purine and pyrimidine. • Mnemonic: • CUT pyr (pie) from Pur A G (ag is gold)

34. Nucleic Acids

35. Nucleic Acids

36. Nucleic Acids • RNA: contains A, G, C but has U instead of T

37. Nucleic Acids • Key things to keep in mind for the Exam: • The purine and pyrimidines bases are on the inside, while the phosphate and deoxyribose units are on the outside of the helix. • Hydrophobic and van der Waals interactions between adjacent base pairs contributes significantly to the stability of the helix. • The two chains are held together by hydrogen bonds • DNA and RNA are read 5’3’ • Your answers for questions relating to Nucleotides should be in 5’3’ • Ex: What is the transcript of this DNA sequence? • a-t-t-g-c-a-g-g-c-c-t-t-a-a-t-g

38. Techniques Based on Differences in Size Dialysis Proteins can be separated from small molecules by dialysis through a semi permeable membrane Gel Filtration (molecular sieve chromatography) Used to separate small molecules from large molecules Small molecules are slowed, caught in the bead (OPP of Gel Electrophoresis) Ultracentrifugation This is used to separate proteins of different sedimentation coefficients. A high molecular weight molecule will sediment faster and diffuse slower than a lower molecular weight molecule of the same density Biopolymer Analysis

39. Techniques Based on Differences in Size Dialysis Biopolymer Analysis

40. Techniques Based on Differences in Size Gel Filtration (Exclusion Chromatography) Biopolymer Analysis

41. Techniques Based on Differences in Size Gel Electrophoresis This technique is used to separate proteins, RNA, DNA and Carbohydrates Large molecules will not move as fast as smaller molecules (sieving effects of gel) In native gel electrophoresis where proteins are not denatured and subunits stay together, the charge of the protein will come into play in the separation Sickle cell anemia and sickle cell trait are diagnosed by a gel electrophoresis method SDS-PAGE Separate proteins based on MW alone Proteins denatured and strongly negative Plot log MW vs. Relative Mobility Biopolymer Analysis

42. Techniques Based on Differences in Size Gel Electrophoresis Biopolymer Analysis

43. Techniques Based on Differences in Size Gel Electrophoresis If an antibody-secreting cell — called a plasma cell — becomes cancerous, it grows into a clone secreting a single kind of antibody molecule. The image shows — from left to right — the electrophoretic separation of: 1. normal human serum with its diffuse band of gamma globulins 2. serum from a patient with multiple myeloma producing an IgG myeloma protein 3. serum from a patient with Waldenström's macroglobulinemia where the cancerous clone 4. secretes an IgM antibody 5. serum with an IgA myeloma protein Biopolymer Analysis

44. Techniques Based on Differences in Charge Ion Exchange Chromatography Negatively charged molecules will bind to columns of positively charged beads (DEAE-Sepharose [diethylaminoethyl-Sepharose]) Positively charged molecules will bind to columns of negatively charged beads (CM-Sepharose [carboxymethyl- Sepharose]) Proteins can be eluted with increasing salt concentrations (Na+ Cl-) because the salt ions compete with the charged groups for binding to the charged column Biopolymer Analysis

45. Techniques Based on Differences in Charge Isoelectric Focusing pI = pH at which the protein has a net ZERO charge Gels containing polyampholytes of differing pHs are prerun to set up gradients of pH, then samples are loaded and electrophoresed until they reach the pH that is equal to their pI ISOFORMS of proteins can be identified by this (differ by single amino acids) Biopolymer Analysis

46. Techniques Based on Differences in Affinity Affinity Chromatography A very specific way to separate proteins or other molecules that bind to or are bound by known molecules Create a column where a specific ligand is conjugated to beads (like Sepharose or agarose) The sample is run through the column, unbound material washed away, and the column is eluted either by competition (for isolating a receptor on a ligand column...elute with excess ligand) or by disrupting binding (for binding that requires divalentcations, chelate these with EDTA) Biopolymer Analysis

47. Techniques Based on Differences in Affinity Affinity Chromatography For example, the antibodies in a serum sample specific for a particular antigenic determinant can be isolated by the use of affinity chromatography Biopolymer Analysis

48. Techniques Based on Differences in Affinity Biopolymer Analysis

49. Identification Using Specific Antibodies ELISA (enzyme linked immunoabsorbant assay) Allows QUANTIFICATION of a specific protein in a mixture http://www.biology.arizona.edu/immunology/activities/elisa/technique.html (animation) Western Immunoblotting Protein mix separated by SDS page then transferred to a membrane support which is incubated with a specific antibody to the protein of interest Then mixed with a secondary antibody that recognized the first antibody (this antibody is radiolabeled) Allows for SPECIFICITY of a specific protein in a mixture http://www.biology.arizona.edu/immunology/activities/western_blot/west1.html Biopolymer Analysis

50. How would you determine the native molecular weight of a protein? Biopolymer Analysis