Exam I

Question 1

1. proteins are least soluble when the net charge is __________?
2. How would you adjust the pH of your solution to accomplish this?
3. This is relevant to what purification technique?

Answer 1

1. zero
2. adjust pH to isoelectric point (pH=pka)
3. salting out, to elute the remaining polar group (usually the desired protein)

Question 2

ion exchange chromatography is best used when the proteins in a mixture for purification ______________

Answer 2

have a big difference in net charge

Question 3

in ion exchange chromatography, what is the name of the chemical attachment to the matrix which serves as the anion exchanger?

Answer 3

DEAE (diethylaminoethyl - positively charged)

Question 4

in ion exchange chromatography, what is the name of the chemical attachment to the matrix which serves as the cation exchanger?

Answer 4

CM (carboxymethyl - negatively charged) CH2—COO^-

Question 5

what is meant by the term “salting in”

Answer 5

the phenomenon of how solubility of a protein at low salt concentrations increases as salt is added because the additional salt shields the protein’s own ionic charges such that the protein doesn’t fold in on itself

Question 6

what is meant by the term “salting out”

Answer 6

when the salt concentration increases beyond the point of salting in, the salt ions then take up solvating water molecules such that the protein has to group together to get solvated (less surface area of solvation for a precipitated protein than for non precipitated) Essentially, the salt out-competes the protein for solvation.

When doing this technique, the salt concentration is usually adjusted to just below the precipitation point of your desired protein.

Question 7

why is affinity chromatography the most powerful chromatography purification technique?

Answer 7

because it exploits the target proteins unique biochemical properties rather than smaller differences like size, polarity, charge, etc.

Question 8

• what do 2-mercaptoethanol or another type of mercaptan do?
• What are they commonly used for?
• The resulting free sulfhydryl groups are then ___1______, usually by treatment with_______2____, to prevent the re-formation of disulfide bonds through ________3____

Answer 8

• they break a disulfide bond to separate subunits of a protein because they themselves contain -SH groups.
• It is used in protein sequencing and SDS-PAGE to separate the subunits of a protein bonded by a disulfide bond. The SDS alone is not going to break this bond.
• 1. alkylated 2. iodoacetate 3. oxidation

Question 9

In SDS-PAGE, the relative mobilities of proteins vary approximately ________ with the logarithm of their _______ .

Answer 9

linearly with the log of their molecular masses

Question 10

What does the mathematical graph for the proteins purified in SDS -PAGE look like in shape and what terms denote the X and Y axes?

Answer 10

Question 11

About half of the 20 amino acids are called essential because

Option A: they are essential for the synthesis of pyrimidines.

Option B: they are essential for nitrogen metabolism.

Option C: they are essential for the synthesis of purines.

Option D: our bodies cannot synthesize them and therefore they need to be present in our diet.

Option E: our bodies need them for protein synthesis.

Answer 11

Option D: our bodies cannot synthesize them and therefore they need to be present in our diet.

Question 12

IEF refers to

Answer 12

isoelelectric focusing

Question 13

what is isoelelectric focusing

Answer 13

when proteins in an electric field that has a gradually increasing pH travel to the point on the field corresponding to their pI

it is often used in conjunction with SDS-PAGE in the perpendicular direction

Question 14

Question NumberQ 1:

Ribosomes use L amino acids to synthesize proteins.These amino acids are called “L” because

Option A: they are all (R)-amino acids.

Option B: they turn polarized light to the left.

Option C: they have a configuration of groups around the Cα that can be related to the configuration of groups around the asymmetric carbon in L-glyceraldehyde.

Option D: they are all (S)-amino acids.

Option E: they are chiral.

Answer 14

Option C: they have a configuration of groups around the Cα that can be related to the configuration of groups around the asymmetric carbon in L-glyceraldehyde.

Question 15

What is an Isopeptide bond

Answer 15

An amide linkage between an α-carboxylate group of an amino acid and the ε-amino group of Lys,

or between the α-amino group of an amino acid and the β- or γ-carboxylate group of Asp or Glu

Question 16

An amide bond between a side-chain carboxylate and an α-amino group is also called a(n)

Option A: isopeptide bond.

Option B: disulfide bond.

Option C: ester bond.

Option D: glycosidic bond.

Option E: anhydride bond.

Answer 16

Option A: isopeptide bond.

Question 17

T/F: Amino acid derivatives play an important role in various biological processes.

Answer 17

True lol duh

Question 18

T/F:

The pK values of the ionizable groups of amino acids may be altered when the amino acid is part of a polypeptide.

Answer 18

True

Question 19

How do you convert mL or L to moles?

*(if you have to figure out an equation with A^-/HA, then you may need to put those quantities in moles form and this is how you can do that)

Answer 19

Question 20

T/F: Amino acids may be covalently modified after they have been incorporated into a polypeptide.

Answer 20

True

Question 21

_____________is the only amino acid of the 20 standard amino acids that lacks a primary amine.

Answer 21

proline

Question 22

A ________bond is a CO-NH linkage that is formed between amino acids.

Answer 22

peptide

Question 23

The polymerization of amino acids to form a polypeptide can be represented as a _______ reaction.

Answer 23

condensation

Question 24

Proteins are molecules that contain one or more __________ chains

Answer 24

polypeptide

Question 25

T/F: In aqueous solution amino acids are rarely found in the neutral, unionized form.

Answer 25

False

Question 26

Lysine has pK values of 2.16, 9.06, and 10.54, for its carboxyl, α-amino, and ℰ-amino groups, respectively. What is its estimated pI? Option A: 9.06 Option B: 9.80 Option C: 5.66 Option D: 6.85

Answer 26

Option B: 9.80

Question 27

All amino acids derived from proteins have the _________ stereochemical configuration.

Answer 27

S (as in R and S) because S corresponds with L and R corresponds with D

Question 28

T/F: The Fischer convention is well suited for describing molecules with multiple chiral centers.

Answer 28

False

Question 29

Glutathione plays a role in \_\_\_\_\_\_\_\_\_

Answer 29

cellular metabolism

Question 30

T/F: Thyroxine is an iodine-containing thyroid hormone.

Answer 30

true

Question 31

T/F: Many of the standard amino acids are chemically modified before incorporation into polypeptide chains.

Answer 31

False, it happens after polypeptide formation

Question 32

T/F: The amino and carboxyl groups at the N- and C-termini of polypeptides can be chemically modified.

Answer 32

True (isopeptide bonds)

Question 33

Which amino acid can be converted to aspartic acid under acidic conditions? Option A: Threonine Option B: Arginine Option C: Glutamic acid Option D: Asparagine

Answer 33

Option D: Asparagine

Question 34

What is the main difference between gel filtration and gel electrophoresis.

Answer 34

What is the main difference between gel filtration and gel electrophoresis.

Question 35

why do you need to reduce a disulfide bonds during Sanger sequencing?

Answer 35

residues in polypeptides that are “knotted” with disulfide bonds may not be accessible to all the enzymes and reagents for sequencing

Question 36

what's so special about mercapto groups?

Answer 36

2-mercaptoethanol or another mercaptan (compounds that contain an —SH group so they can "take over" the disulfide bonds to break them

Question 37

Water solvates both positive and negative ions.

Answer 37

true

Question 38

Amphiphiles or amphipathic molecules are molecules that have both polar and nonpolar segments.

Answer 38

true

Question 39

Which description best characterizes the length of a hydrogen bond between water molecules? Option A: About the same length as a calculated O-H van der Waals distance Option B: Shorter than a covalent O-H bond Option C: Longer than a covalent O-H bond, but shorter than the calculated van der Waals distance Option D: About the same length as a covalent O-H bond

Answer 39

C

Question 40

Which of the properties listed is NOT true of liquid water? Option A: Liquid water is less dense than ice. Option B: Liquid water tends to form hydrogen bonded rings of three to seven molecules. Option C: Liquid water consists of a rapidly fluctuating, three-dimensional network of hydrogen-bonded molecules. Option D: Liquid water is only 15% less hydrogen bonded than ice at 0o C.

Answer 40

A

Question 41

Which statement best explains why nonpolar substances have low solubility in water? Option A: They have strong mutual attractions. Option B: They cannot form hydrogen bonds to water. Option C: They are much larger than water. Option D: They are much denser than water.

Answer 41

B

Question 42

The negative of the log[H+] is defined as

Answer 42

pH

Question 43

T/F The acids listed in Table 2.4 are known as weak acids because they are only partially ionized in aqueous solution (K \< 1)(K \< 1).

Answer 43

T

Question 44

T/F: The ionic mobilities of H+ and OH- are much greater than that of other ions.

Answer 44

T

Question 45

T/F: The value of Kw is 10¹⁴ M2 at 25°C.

Answer 45

F: because it should be 10^-14

Question 46

T/F: The buffering capacity of a weak acid is greatest when pH = pKa.

Answer 46

True

Question 47

What is the concentration of hydrogen ions at pH 3? Option A: 0.3 M Option B: 103 M Option C: 10-3 M Option D: 3 M

Answer 47

C pH=3 pH= -log[H⁺] 3= -log[H⁺] 3= -log10^-3 So, H⁺=10^-3

Question 48

What is the concentration of hydroxide ions at pH 10? Option A: 10^-4 M Option B: 10¹⁰ M Option C: 10^-10 M Option D: 10⁴ M

Answer 48

A _use pH=-log[H⁺] 10= -log[H⁺] 10= -log 10^-10, so [H⁺] = 10^-10 _{then use} K_w=[H⁺][OH^-] K_w=[10^-10][OH^-] *_but* K_w *_{also =} *10^-14 _{a constant from literature} so, 10^-14=[10^-10][OH^-] *_{use rule: when you multiply numbers with exponents, you add exponents} **_{so, -14= -10 + (-4)}* [OH^-] =10^-4

Question 49

What is the pH of a solution made by mixing equal volumes of 1 M sodium acetate and 1 M acetic acid? (The pK of acetic acid is 4.76.) Option A: 4.76 Option B: 5.76 Option C: 3.76 Option D: 1

Answer 49

A because when A-/HA = 1 log1 = 0 and so pH=pk + 0

Question 50

Which statement about the pK is not true? Option A: The pK is equal to the pH at the midpoint of the titration. Option B: The pK is equal to the pH at the maximum slope of the titration curve. Option C: The pK is equal to the pH when [A-] = [HA]. Option D: The pK is equal to the pH at the maximal buffering capacity.

Answer 50

B

Question 51

Which of the following statements is ***_not_*** true? Option A: Ammonium hydroxide (pK = 9.25) is an effective buffer against base at pH 9.75. Option B: Acetic acid (pK = 4.76) is an effective buffer against acid at pH 3.76. Option C: Acetic acid (pK = 4.76) is an effective buffer against acid at pH 5.76. Option D: Ammonium hydroxide (pK = 9.25) is an effective buffer against base at pH 8.25.

Answer 51

B ## Footnote A weak acid is in its useful buffer range within one pH unit of its pK so we don't know why this is the right answer

Question 52

The pK of acetic acid is 4.76. Which statement is true at pH 5.0? Option A: [A-] \> [HA] Option B: [A-] = [HA] Option C: [A-] \< [HA] Option D: [A-] \>\> [HA]

Answer 52

[A-] \> [HA] 5=4.76+logA-/HA ^{_{(substituting henderson hasselbach)}} .24 = logA-/HA .24=log 10^.24 A-/HA= 10^.24/1, ^{_{so there is more A- than HA}}

Question 53

The pK values for phosphoric acid are 2.15, 6.82, and 12.38. At pH 12.38, [H2PO4-] = [HPO42-].

Answer 53

False

Question 54

The pK values for phosphoric acid are 2.15, 6.82, and 12.38. At pH 6.82, [H2PO4-] = [HPO42-].

Answer 54

True

Question 55

At the first horizontal inflection point in the titration curve of phosphoric acid, which statement is true? Option A: [H3PO4] = [H2PO4-] Option B: [H2PO4-] = [HPO42-] Option C: [H3PO4] \> [H2PO4-] Option D: [H2PO4-] \< [HPO42-]

Answer 55

A horizontal inflection point = same as midpoint

Question 56

Consider a dialysis procedure in which an aqueous solution is separated from pure water by a membrane that is permeable to both water and solutes. Which statement is false? Option A: The tendency of solutes to diffuse from a region of high concentration to a region of lower concentration is thermodynamically favored. Option B: Dialysis cannot separate molecules based upon their relative sizes. Option C: The membrane can be fine-tuned to selectively allow large molecules instead of smaller molecules to diffuse through it. Option D: The random movement of molecules, called diffusion, proceeds only until the concentration is the same on each side of the dialysis membrane.

Answer 56

Option B: Dialysis cannot separate molecules based upon their relative sizes.

Question 57

From the pK’s in table 2-4, considering all other factors equal, which weak acid/conjugate base pair will give the best buffer for a pH of 4.3? Option A: succinate-/ succinate2- Option B: succinic acid/succinate- Option C: oxalic acid/oxalate- Option D: acetic acid/acetate-

Answer 57

B: succinic acid/succinate- because the midpoint of the PKas of Succenic/succinate is 4.925 which is closer to the given pH

Question 58

Ortholog def

Answer 58

Homologous proteins with the same function expressed in different species (e.g. the cytochrome c) (Homologous: evolutionarily related) \*To acheive this, you can only have variation in hypervariant regions or conservative substition. No invariant substitution, otherwise they would not be orthologs

Question 59

1. How does the sequence of the protein of cytochrome C affect its ability to bind to the peripheral membrane? 2. What is the nature of this bond?

Answer 59

1. Cytoplasmic face of a phospholipid membrane is negatively charged, so in order to connect this protein to the membrane, you have to have a positively charged protein, which is why there are a lot of K’s in the protein (K = lysine) 2. ionic interaction (non-covalent)

Question 60

what are the 5 factors that are important to consider when purifying proteins?

Answer 60

(1) pH (2) Temperature. ^{_{Disruption of non-covalent bonds possible (denaturing).}} (3) Presence of degradative enzymes. ^{_{(when you burst your cell to access the protein of interest, you could be bursting lysosomes with enzymes so you have to account for that)}} (4) A***d***sorption to surfaces. ^{_{Sticking to surfaces as opposed to penetrating the surface in absorption. Some containers can have residual plolarity or charge you have to eliminate prior.}} (5) Long-term storage.

Question 61

Dansyl chloride is used for: Option A: polypeptide cleavage on the C side of Met. Option B: reduction of disulfide bonds between cysteine residues. Option C: polypeptide cleavage on the C side of Glu. Option D: amino-terminal determination. Option E: polypeptide cleavage on the C side of Lys or Arg.

Answer 61

Option D: amino-terminal determination.

Question 62

2-Mercaptoethanol is used for: Option A: polypeptide cleavage on the C side of Met. Option B: polypeptide cleavage on the C side of Glu. Option C: reduction of disulfide bonds between cysteine residues. Option D: polypeptide cleavage on the C side of Lys or Arg. Option E: amino-terminal determination.

Answer 62

Option C: reduction of disulfide bonds between cysteine residues.

Question 63

Chymotrypsin is used for: Option A: polypeptide cleavage on the C side of Trp, Tyr or Phe. Option B: polypeptide cleavage on the C side of Glu. Option C: polypeptide cleavage on the C side of Lys or Arg. Option D: reduction of disulfide bonds between cysteine residues. Option E: polypeptide cleavage on the C side of Met. F Option F: amino-terminal determination.

Answer 63

Option A: polypeptide cleavage on the C side of Trp, Tyr or Phe.

Question 64

T/F:Electrospray ionization (ESI) is an ionization technique used in the sequencing of peptides by mass spectrometry; it involves vaporization by heating followed by ionization via bombardment with electrons.

Answer 64

FALSE: because ESI and mass spec are different, def is for mass spec only

Question 65

What is the only amino acid that that does not have an "S" configuration?

Answer 65

cysteine (but it's still "L)

Question 66

for this exam, is histadine protonated or deprotonated at pH of 7?

Answer 66

deprotonated (so it's neutral at pH 7) \*this is because it's borderline b/c it has pKa of 6

Question 67

T/ F: The aromatic residues phenylalanine, tyrosine and tryptophan are the three amino acids responsible for proteins high absorption at the 280 nm wavelength, therefore measuring a sample's UV absorbance at 280 nm gives an accurate measurement of its protein content.

Answer 67

False

Question 68

T/F: a protein is most soluble at it's isoelectric point

Answer 68

false

Question 69

Enzymes that catalyze the hydrolysis of N- or C-terminal residues are known as

Answer 69

exopeptidase e.g. Edman's reagent

Question 70

\_\_\_\_\_\_\_\_\_uses a stable pH gradient to separate proteins.

Answer 70

isoelectric focusing

Question 71

The amino acid sequence of a polypeptide can be determined through repetitive cycles of \_\_\_\_\_\_\_\_

Answer 71

edman degredation

Question 72

T/F: The buffering capacity of a weak acid is greatest when pH = pKa.

Answer 72

True

Question 73

The pK of ammonia is 9.25. What is the effective buffering range? Option A: pH = 9.25 Option B: pH = 8.25-10.25 Option C: pH = 9.15-9.35 Option D: pH = 7-13

Answer 73

Option B: pH = 8.25-10.25

Question 74

\_\_\_\_\_\_\_\_\_is commonly used for salting out proteins because of it's high solubility in water

Answer 74

ammonium sulfate

Question 75

Question 76

Which statement about the pK is **_not_** true? Option A: The pK is equal to the pH at the midpoint of the titration. Option B: The pK is equal to the pH when [A-] = [HA]. Option C: The pK is equal to the pH at the maximal buffering capacity. Option D: The pK is equal to the pH at the maximum slope of the titration curve.

Answer 76

Option D: The pK is equal to the pH at the maximum slope of the titration curve.

Question 77

Which statement about insulin is correct? Option A: Insulin is composed of two polypeptides, the A chain and the B chain. Option B: Insulin contains an intrachain disulfide bond. Option C: Insulin contains interchain disulfide bonds. Option D: The A chain and the B chain of insulin are encoded by a single gene. Option E: All of the above are correct.

Answer 77

E. All of the above

Question 78

Does metabolic alkalosis (excess of HCO₃-) increase or decrease CO2 in the blood?

Answer 78

In class he said it increases it. This is only true if it's implied that H+ also increases, which will push the equation to the left. But in reality, that is the slower mechanism. The faster mechanism is compensatory hypoventilation, which will increase CO² on it's own, thereby pushing the equation to the right and increasing H₂CO₃ (cabonic acid). The pH is determined by the ratio of HCO₃-/H₂CO_3. (because that's the A-/HA component to hendersen hasselbach for blood. Normal is 20/1 ratio of HCO₃-/H₂CO_3. \*For the test, assume that H⁺ is increased along with HCO₃-, pushing the equation to the left.

Question 79

what's the difference between a dipole-induced dipole and a permanent dipole?

Answer 79

Question 80

In sequencing a protein (after cleavage of disulfide bond) you first complete chemical and enzymatic cleaving followed by Edman degredation to identify the sequence of your peptide fragments. What do you do next?

Answer 80

You need to figure out where those sequenced fragments fall within the whole peptide. To do that you do an additional round of chemical and enzymatic cleaving with reagents of different cleavage specificity followed by Edman's sequencing, such that you have overlapping sequences. You use these sets of overlapping sequences to decipher the original peptide sequence.

Question 81

What is the final step in protein sequencing?

Answer 81

Find where the disulfide bond is. To do this you repeat fragmentation of your original peptide, except this time, you do not break the disulfide bonds. This allows you to identify the Cys-containing sequence involved in the disulfide linkages. After isolating a disulfide-linked polypeptide fragment, the disulfide bond is **again** cleaved and alkylated/capped and the sequences of the two peptides are determined. Finally, compare the sequence of each subunit to the subunit sequences you arrived at before. And this gives you a complete sequence of your original protein with 2 polypeptide subunits.

Question 82

During polypeptide cleavage into segments (3rd step in sequencing), what would interfere with the cleavage of a given amino acid?

Answer 82

If the next AA to the ***right*** (C terminal side) of the target AA is proline, cleavage will not occur. \*true for all enzyme scissors except for Endopeptidase V8, which doesn't care about Pro

Question 83

What is the first step in protein sequencing and how do we do it?

Answer 83

First step is to identify the terminal ends using the N terminus, which tells us how many subunits there are. We can accomplish this using fluoresent dansyl chloride or by doing a single Edman degredation. \*Lysine will give a false positive for protein N terminus because of it's free primary amine group

Question 84

How does a mercaptan accomplish its task

Answer 84

reduction of disulphide bond (H addition) using 2 molecules of the reagent. The resulting product is 2 cleaved polypeptides and a mercaptan reagent that now has a disulfide bond

Question 85

What is the reagent used to cap the free SH ends after cleavage of disulfide bond, called alkylation

Answer 85

iodoacetate see pic for resulting products

Question 86

Polypeptides that are longer than \_\_\_\_\_\_\_residues cannot be directly sequenced and must be cleaved

Answer 86

40 to 100

Question 87

Endopeptidases and exopeptidases cleave a peptide bond by which of these processes? 1. oxidation 2. reduction 3. hydrolysis

Answer 87

3. hydrolysis

Question 88

The digestive enzyme \_\_\_\_\_\_\_\_\_\_has the greatest specificity and is the most valuable member of the arsenal of endopeptidases used to fragment polypeptides.

Answer 88

trypsin

Question 89

How does cyanogen bromide work?

Answer 89

It works on Methionine because the Sulphur from that side chain will attack the cyano group, creating an excellent leaving group. This loss of electrons causes a ring structure to form on the C side of Met. Hydrolysis is what cleaves the peptide bond connecting to the next AA, yielding a peptidyl homoserine lactone and the remaining peptide.

Question 90

Trypsin cleaves \_\_\_\_\_

Answer 90

the positively charged AAs arginine and lysine (remember: it's #1 so it gets an A+ twice)

Question 91

Chymotrypsin cleaves \_\_\_\_\_\_

Answer 91

the aromatics: FWY Phe, Trp, Tyr,

Question 92

How does Edman degredation work? How does its reagent work?

Answer 92

Repeated cycles of N residue removal using PITC Reaction steps: 1. **PITC** reacts with the polypeptide to form a PTC-polypeptide or **PTC adduct** 2. **anhydrous trifluoroacetic acid** is used to catalyze the formation of a ring structure that is essentially half PITC and have N residue, thereby cleaving it from the original peptide. This ring structure is called **Thiazolinone derivative** 3. Acid catalyzed hydrolysis yields the final **PTH-amino acid derivative** (more stable form). So, overall you go from **PITC -\>PTC-\>PTH**

Question 93

What is neutral drift?

Answer 93

When the random nature of mutational processes will in time change a protein in ways that do not significantly affect its function. Hypervariable residues undergot neutral drift. Cytochrome C

Question 94

How does the invariant residue relate to the protein function.

Answer 94

Through evolution we can see that if only 1 proteins has ever been successfully substituted a different protein at a particular spot in the sequence it must mean that location in the sequence is vital to some essential function of the protein.

Question 95

what is a domain?

Answer 95

evolutionarily conserved segments of a protein of about 40-200 residues. domains that are 40% identical usually have the same function. less than 25% identical - different roles

Question 96

What is a paralog?

Answer 96

Two independently evolving (meaning, adopting a new function) genes that are derived from the same duplication event. They are homologus but not orthologous.

Question 97

The human genome contains the relics of globin genes that are not expressed and are inactive. These can be considered the dead ends of protein evolution and are called \_\_\_\_\_\_\_\_

Answer 97

pseudogenes \*A new gene from a duplication event has only a limited time to evolve a new functionality that provides a selective advantage to its host before it is inactivated through mutation

Question 98

The plot for a given protein is essentially linear, indicating what?

Answer 98

that its mutations accumulate at a constant rate over a geological time scale.

Question 99

a protein that is highly conserved is said to have more invariant or hypervariable regions

Answer 99

invariant regions

Question 100

T/F: The genes for highly expressed proteins appear to evolve more slowly than the genes for rarely expressed proteins.

Answer 100

true

Question 101

T/F: Domains occur in several other proteins in the same organism and may be repeated numerous times within a given protein

Answer 101

True

Question 102

T/F: he smaller a protein is, the faster it will be moved through SDS-PAGE

Answer 102

true

Question 103

In gel filtration chromatography the \_\_\_\_\_\_\_\_protein travels down first, whereas in gel electrophoresis, the \_\_\_\_\_\_\_\_\_\_\_\_protein travels first

Answer 103

larger - gel chrom smaller - gel electro

Question 104

How is salt used to elute protein of interest in hydrophobic interaction chromatography?

Answer 104

salt removes the shielding on protein so that its hydrophobic domain is exposed, maximizing th interaction with stationary phase. As you decrease the salt concentration the more water is free to interact with the polar groups, which decreases their attraction to the mobile phase

Question 105

How do you use coomassie brilliant blue?

Answer 105

The goal is to get the coommassie brilliant blue molecule to make a covalent bond with your protein of interest, which happens in acidic conditions (pH 1-2). This shifts the compound's absorbance frequency to the visible light spectrum. When that happens, your protein can be measured by the brightness of the blue color. Titrations of the same protein of known concentration can then be used for comparison to determine the concentration of protein you had to start with. \*denaturing of proteins d/t the low pH will not interfere with this measurement

Question 106

How is the concentration of a protein detected by ELISA?

Answer 106

by color - either the detectible product reacts with a solution to form a color within the visible light range, whose intensity can be quantified, or if the product is aromatic you can run a UV spectroscopy test on it, using the UV light range

Question 107

Polypeptides absorb strongly in the ultraviolet (UV) region of the spectrum (λ = 200 to 400 nm, **especially 280**) largely because their aromatic side chains (those of Phe, Trp, and Tyr) have particularly large \_\_\_\_\_\_\_\_\_\_in this spectral region

Answer 107

extinction coefficients

Question 108

In what spectrum does the Bradford assay (coommassie brilliant blue) operate?

Answer 108

595 nm (visible light spectrum)

Question 109

You are using ammonium sulfate to purify protein Q (pI = 5.0) by salting out from a solution at pH 7.0. How should you adjust the pH of the mixture to maximize the amount of protein Q that precipitates?

Answer 109

Lowering the pH from 7.0 to 5.0 would promote the precipitation of protein Q because the protein will be least soluble when its net charge is zero (when pH = pI)

Question 110

what does SDS stand for

Answer 110

sodium dodecyl sulfate

Question 111

Edman degradation can be used to Option A: separate the subunits of a multi-subunit protein. Option B: identify the C-terminal amino acid of a polypeptide. Option C: cleave disulfide bonds within a protein so that the individual polypeptides can be separated. Option D: cleave a protein at specific sites. Option E: identify the N-terminal amino acid of a polypeptide.

Answer 111

Option E: identify the N-terminal amino acid of a polypeptide.

Question 112

def of gene duplication

Answer 112

similar proteins arise through gene duplication, an aberrant genetic recombination event in which one member of a chromosome pair acquires both copies of the primordial gene

Question 113

Identify the least conservative amino acid substitution, assuming that these two residues occur at the same position in two homologous proteins. Option A: Glu --\> Lys Option B: Glu --\> Ala Option C: Glu --\> Leu Option D: Glu --\> Asp

Answer 113

Option A: Glu --\> Lys because the charges are opposite?

Question 114

Simple molecules condense to form more complex molecules called \_\_\_\_\_\_\_

Answer 114

polymers

Question 115

define complementarity of molecules

Answer 115

reciprocal pairing in which one member of a pair of molecules determines the identity and orientation of the other member e.g hydrogen bonding and DNA base pairing. This principle is a theory as to how we transitioned from randomly paired molecules to systems of molecules that were organized and could replicate.

Question 116

what are the advantages to compartmentation

Answer 116

1. protection from environment 2. maintain high local concentrations of components (particularly ions) that would otherwise diffuse away. This concentration gradient allows for efficient chemical reactivity 3. has evolved to contain more complex machinary in the cell (such as enzymes and metabolic pathways) which makes the organism more stable in environments with limited resources.

Question 117

What are the disavantages of compartmentation?

Answer 117

1. organisms are not well suited to a variety of habitats. 2. exchange of nutrients and waste is inefficient because of the lipid bilayer nature of the compartments - they only diffuse lipid soluble substances 3. rapid growth rates not possible

Question 118

What does a thioester look like?

Answer 118

Question 119

What does it mean to say "linkage"

Answer 119

Question 120

What is an amido group

Answer 120

Question 121

Visualize the various phosphate groups and linkages possible

Answer 121

Question 122

T/F: If energy goes into a system, then the reverse reaction will mean energy is released

Answer 122

True

Question 123

What best differentiates eukaryotes from prokaryotes and what are the other differences.

Answer 123

In order of importance: 1. Euks have **membrane bound organelles** 2. Euks **compartmentalization** of outer layer as well (lipid bilayer plasma membrane) 3. Euks have **neucleus** 4. **areobic respiration** is required and euks have complex machinary to meet that need 5. Euks are **large** and grow/multiply slowly -size is 1k to 1 million times larger than prok 6. **Transcription and translation** happens in different compartments. Transcription and translation happens in the cytoplasm for proks so it's simultaneous. 7. Almost all Prokaryotes are unicellular, whereas only some euks are unicellular

Question 124

T/F: Different metabolic functions are believed to be carried out in different regions of the cytoplasm in prokaryotes

Answer 124

True

Question 125

T/ F: •The eukaryotic genetic material includes features that suggest an archaebacterial origin.

Answer 125

True

Question 126

T/F Most living matter consists of a relatively large number of chemical elements.

Answer 126

False most of living matter is bacteria which is relatively smaller number of chemical elements

Question 127

T/F The diameter of a typical prokaryotic cell is about 1-10 micrometers whereas the diameter of a typical eukaryotic cell is about 10-100 micrometers.

Answer 127

True

Question 128

\_\_\_\_\_\_ is the science of biological classification.

Answer 128

taxonomy

Question 129

T/F: The best way to determine phylogenetic relationships between organisms is by comparing morphological differences.

Answer 129

False

Question 130

T/F: A process with a large negative ∆G will proceed rapidly.

Answer 130

False - not necessarily

Question 131

Which statement about a chemical reaction at equilibrium is false? Option A: ∆G = 0 Option B: Forward and reverse reactions proceed, but the concentrations of reactants and products do not change. Option C: ∆H = T∆S Option D: The concentration of products equals the concentration of reactants.

Answer 131

Option D: The concentration of products equals the concentration of reactants.

Question 132

Change in enthalpy (∆H) is best defined as Option A: none of the above Option B: the heat transferred at constant pressure. Option C: the sum of heat absorbed and work done. Option D: the measure of disorder in a system. Option E: the pressure change at constant temperature.

Answer 132

Option B: the heat transferred at constant pressure.

Question 133

T/F: entropy and enthalpy of a system are inverseley related

Answer 133

if entropy of a system decreases then enthalpy change is positive

Question 134

K ratio represents (3 things)

Answer 134

Deprotonated/protonated Unbound/bound Products/Reactants

Question 135

How would you compare bond angles of water to methyl group?

Answer 135

Methyl: 109.5° H20: 104.5°

Question 136

1. What is an amphiphilic molecule and what's an example? 2. How do amphiphiles interact with an aqueous solvent?

Answer 136

1. Most biological molecules have both polar and nonpolar segments and are therefore simultaneously hydrophilic and hydrophobic. 2. Water tends to hydrate the hydrophilic portion of an amphiphile, but it also tends to exclude the hydrophobic portion. Amphiphiles consequently tend to form structurally ordered aggregates. For example, **micelles**

Question 137

Describe the main property of water as a solvent

Answer 137

1. The oxygen atom with its unshared electrons carries a partial negative charge and the hydrogen atoms each carry a partial positive charge, leading to electrostatic attractions between the dipoles of water leading to **hydrogen bonding** 2. tetrahedral geometry causes water to expand and have an open structure when in ice form, this makes it so that ice is actually less dense than water. Unlike other liquids that cannot hydrogen bond 3. its boiling point is higher than that of other molecules of similar mass d/t comparative difficulty in breaking hydrogen bonds 4. water consists of rapidly fluctuating three-dimensional networks of clusters of hydrogen bonded H20 molecules. up to seven membered rings are possible in liquid water at any given moment. 5. disordering of water molecules (increase in entropy, delta S) drives water processes

Question 138

Why do non-polar solutes aggregate in water?

Answer 138

The reason that non-polar solutes aggregate in water is that aggregation decreases the surface area around which water creates a cage (ie an orientation of water molecules around the solute such that they are maximizing hydrogen bonding.) This cage formation constitutes an order (and unfavorable free energy of hydration) insofar as the ways that they can hydrogen bond become limited, decrease in entropy (entropy is at it’s greatest in bulk water). So by limiting the area available for caging, they are reducing order, which is favored. This aggregation is why water and oil don’t mix.

Question 139

T/F: Hydrogen bonds are structurally characterized by an H···A distance that is at least 0.5 Å shorter than the calculated van der Waals distance (the distance of closest approach between two nonbonded atoms).

Answer 139

True

Question 140

Does solvating (aka hydrating) an ion or polar molecule constitute an increase or decrease in entropy?

Answer 140

A decrease in entropy because the water becomes ordered in forming a hydration shell favorable free energy of hydration

Question 141

–Do functional groups have to ionized to exhibit hydrophilic properties?

Answer 141

no, the bond dipoles of uncharged polar molecules make them soluble in aqueous solutions for the same reasons that ionic substances are water soluble.

Question 142

Define Hydrophobic effect

Answer 142

The tendency of water to minimize its contacts with hydrophobic molecules.

Question 143

\_\_\_\_\_\_\_\_\_ form Micelles and Bilayers

Answer 143

Amphiphiles Whether it becomes a micelle or bilayer or vessicle (which is made up of bilayer) depends on amount of lipid added as well as length of hydrocarbon chain

Question 144

Dialysis T/F: At equilibrium, the concentrations of small molecules are nearly the same on either side of the membrane, whereas the macromolecules remain inside the dialysis bag.

Answer 144

True

Question 145

T/F: mobility of H+ and OH– ions in aqueous solution is much higher than for other ions

Answer 145

True

Question 146

What's the mathematical relationship between concentration of H+ and concentration of OH- ?

Answer 146

–Pure water must contain equimolar amounts of H+ and OH–, so [H+] = [OH–] = (Kw)1/2 = 10–7 M. –Since [H+] and [OH–] are reciprocally related, when [H+] is greater than 10–7 M, [OH–] must be correspondingly less and vice versa. –Solutions with [H+] = 10–7 M are said to be neutral, those with [H+] \> 10–7 M are said to be acidic, and those with [H+] \< 10–7 M are said to be basic.

Question 147

At what pH of aqueous solution will you have an ionic interaction between solute and water?

Answer 147

The pH at which you have a charge for each.

Question 148

You're given a pH of pure water, what equation do you use to figure out the pOH?

Answer 148

K= [H+] [OH-] = 1 x 10^-14 so, in this example, pOH would be 10^-11

Question 149

def of weak acid

Answer 149

only partially ionized in aqueous solution (K\<1)

Question 150

strong acid def

Answer 150

K\>\>1, readily ionized in water

Question 151

Given a pI of 11 and a pH. How do you figure out the charge?

Answer 151

Question 152

T/F: H+ and OH- are in equal concentrations in water at pH 7

Answer 152

true

Question 153

The phosphate and bicarbonate ions in most biological fluids are important buffering agents why?

Answer 153

because they have pKs close to their pHs

Question 154

What happens when the ideal buffering region is passed?

Answer 154

–Above or below this range, the pH of the solution changes rapidly with added base or acid

Question 155

T/F: Not every protein contains all 20 types of amino acids, but most proteins contain most if not all of the 20 types.

Answer 155

True

Question 156

T/F: some proteins are produced as single chain, but become multisubunit chains after activation

Answer 156

true

Question 157

What is the great limitation in the study of primary structure and why does this matter

Answer 157

it doesn’t tell us what the folded protein looks like. Since differences in folding account for different orthologs, this is functionally significant in studying organisms

Question 158

glutamine is \_\_\_\_\_yl glutamic acid is \_\_\_\_\_yl ^in peptide nomenclature

Answer 158

glutamine becoems glutaminyl glutamic acid: glutanyl

Question 159

If enantiomeric molecules are physically and chemically indistinguishable by most techniques, how can we differentiate between them?

Answer 159

Only when probed asymmetrically, for example, by plane-polarized light or by reactants that also contain chiral centers, can they be distinguished or differentially manipulated.

Question 160

In the Fischer system, by convention the configuration of the groups around an asymmetric center is compared to that of glyceraldehyde, a molecule with one asymmetric center. These molecules are said to have the same \_\_\_\_?

Answer 160

relative configuration For almost all AAs L=S and D=R, but because of this convention, cysteine is both L and R because the sulfur group alters the priority of the groups (SH\>OH), but we still liken its configuration to Glyceraldehyde

Question 161

What are the various contributors to the rate of divergence (ie rate at which mutations are accepted into a protein)?

Answer 161

1. **effect of amino acid changes on the protein's function.** e. g. histone H4 is so finely tuned to its function that any changes in its sequence must be compatible with all its binding partners. Whereas when fibrinogen is activated by cleaving of a segment that is then discarded, so clearly that region's sequence matter less which is why its rate of divergence is very high 2. protein's **structural stability.** e. g. a mutation that affected protein folding (or the rate thereof) could affect cell's survival. ^This is why genes that are more highly expressed mutate more slowly than those that are rarely expressed.

Question 162

What is the purpose of analyzing the primary structure of an amino acid?

Answer 162

Helps us see mutational relationships and evolution

Question 163

How does ESI work?

Answer 163

Dry N2 gas promotes the evaporation of solvent from charged droplets containing the protein of interest, leaving gas-phase ions, whose charge is due to the protonation of Arg and Lys residues. The mass spectrometer then determines the mass-to-charge ratio of these ions. The resulting mass spectrum consists of a series of peaks corresponding to ions that differ by a single ionic charge and the mass of one proton)

Question 164

Define ELISA

Answer 164

Enzyme-linked Immunosorbent Assay It is tool used to measure the concentration of a given protein based on the 1:1 relationship of protein to product. By measuring the concentration of the product using absorbance in visible light or UV light, you are measuring the concentration of the protein itself.

Question 166

what amino acid modification does glutathione have?

Answer 166

isopeptide bond formation

Question 167

In general, proteins contain at least \_\_\_\_residues or so; polypeptides smaller than that are simply called peptides.

Answer 167

40

Question 168

what's an inclusion body?

Answer 168

A genetically engineered organism that produces large amounts of a foreign protein often sequesters it in inclusion bodies.

Question 169

What's the limitation of the Fischer system?

Answer 169

It can't encompass molecules with more than one chiral center. e.g. L-threonine can also be called (2S,3R)-threonine.

Question 170

how is the pH of electrophoresis field adjusted to ensure it will work? (in the case of non SDSPAGE electrophoresis)

Answer 170

The pH of the gel is high enough (usually about pH 9) so that nearly all proteins have net negative charges and move toward the positive electrode (bottom) when the current is switched on.

Question 171

SDS-PAGE separates proteins purely by gel filtration effects, that is, according to \_\_\_\_\_\_\_

Answer 171

molecular mass. this occurs because their ionic charge is masked by SDS

Question 172

You have your peptide changes segmented and ready for sequencing, how do you do that?

Answer 172

use edman degredation: ultimately it's the **PTH-amino acid** is what’s measured by gel chromatography because each N residue will have a diff weight

Question 173

how does tandem spec work?

Answer 173

–The first mass spectrometer functions to select and separate the peptide ion of interest from peptide ions of different masses as well as any contaminants that may be present. –The selected peptide ion is then passed into a collision cell, where it collides with chemically inert atoms such as helium. –The energy imparted to the peptide ion causes it to fragment predominantly at only one of its several peptide bonds, yielding one or two charged fragments per original ion. –The molecular masses of the numerous charged fragments produced are determined by the second mass spectrometer.

Question 174

Limitations and advantages of mass spec?

Answer 174

Disadvantages: The sequence of an entire polypeptide can thus be elucidated (although mass spectrometry cannot distinguish the isomeric residues Ile and Leu because they have exactly the same mass, and it cannot always reliably distinguish Gln and Lys residues because their molecular masses differ by only 0.036 D). Advantages: Mass spectrometry can be used to sequence peptides with chemically blocked N-termini (which prevents Edman degradation) and to characterize other posttranslational modifications

Question 175

What is the Bradford assay?

Answer 175

The Bradford protein assay is a time-tested colorimetric assay. When the Bradford reagent (acidified Coomassie Brilliant Blue G-250) binds to proteins, the dye undergoes a color change in the visible spectrum, with the absorbance maximum moving from 470 to 595 nm. The absorbance at 595 nm is then read either in a spectrophotometer or a microplate reader and is directly proportional to the amount of protein bound. The exact protein concentration of the sample is determined by interpolation from a standard curve made by measuring the absorbance of a dilution series of protein standards of known concentrations within the linear response range of the Bradford protein assay. Proteins commonly used as standards include bovine serum albumin (BSA) and bovine γ-globulin (BGG).

Question 176

Bond strengths

Answer 176

covalent: 200+ in kJ per mol^-1 Ionic: 90 (so old she's been around for eons.. I mean ions) Van der Whals * Hydrogen: 20s (age of alcohol consumption) * Dipole-dipole: 10 (D for decem, which means 10) * London Dispersion Forces: \<1