Chapter 3: Protein Structure and Function (part 2) Flashcards
1
Q
where is alpha carbon attached to
A
- carboxyl group (-COOH)
- amino group (-NH2)
2
Q
at pysiologic pH, the amino acid has what?
A
- -COO-
- -NH3+
3
Q
neutral molecule with equal number of + and - charges
A
zwitterion
4
Q
zwitterion is from the word what?
A
German “zwitter” = “hybrid” or “hermaphrodite”
5
Q
what do the two charged groups at the two ends of the amino acid lead to
A
internal proton transfer, forming zwitterions
6
Q
how do you affect the net charge on zwitterions
A
by changing pH
7
Q
pH point at which there is no net charge on zwitterions
A
isoelectric point (pI)
8
Q
pH 7
A
physiological pH
9
Q
zwitterion
A
internal salt
10
Q
what is internal salt
A
product of acid-base reaction between carboxylic acid and amine
11
Q
At the isoelectric point, what will happen to the amino acid?
A
not migrate in an applied electric field
12
Q
zwitterions have higher __ __
A
melting points
13
Q
test done by applying a sample of amino acid to specially treated paper or gel and applying electric field at different pH values
A
electrophoresis
14
Q
amino acid bearing a positive charge will migrate where
A
cathode (-)
15
Q
amino acid bearing a negative charge will migrate where
A
anode (+)
16
Q
weakly basic
A
carboxylate group
17
Q
at an acidic enough pH, what will happen to the carboxylate group
A
protonated creating carboxylic acid
18
Q
weakly acidic
A
ammonium group
19
Q
at a basic enough pH, what will happen to the ammonium group
A
deprotonated creating amine
20
Q
net charge of protonated of carboxylate group
A
+1
21
Q
net charge of deprotonated ammonium
A
-1
22
Q
isoelectric point represent the pH values of what concentration
A
acid = conjugate base
23
Q
pI formula
A
pI= (pKa1 + pKa2) / 2
24
Q
equation which states [HA] = [A-], pH = pKa
A
Henderson-Hasselbalch equation
25
indicates whether an acid is a strong acid or a weak acid
pKa
26
in an amino acid with acidic sidechain, where does positively charged form (+1) dominate
very low pH
27
in an amino acid with acidic sidechain, where does zwitterionic (neutral) form dominate
low pH
28
in an amino acid with acidic sidechain, where does negatively charged form (-1) dominate
intermediate pH
29
in an amino acid with acidic sidechain, where does doubly negatively charged form (-2) dominate
strongly basic pH
30
in an amino acid with acidic sidechain, the isolelectric point should be the average of what
pKa values of two most acidic groups
31
in an amino acid with basic sidechain, where does double positively charged form (+2) dominate
very low pH
32
in an amino acid with basic sidechain, where does positively charged form (+1) dominate
intermediate pH
33
in an amino acid with basic sidechain, where does the zwitterionic form (neutral) dominate
basic pH
34
in an amino acid with basic sidechain, where does the negatively charged form (-1) dominate
strongly basic pH
35
in an amino acid with basic sidechain, the isolelectric point should be the average of what
two most basic groups (pKa2 and pKa3)
36
the pI values of polar acidic amino acids are about what pH?
pH 3
37
analytical method for identifying amino acids by observing their migration as a function of pH under an applied electric field gradient
electrophoresis
38
Electrophoresis steps
1. paper or gel (polyacrylamide) saturated with buffer solution
2. soln. of AA is placed at center of paper
3. electrodes are connected to ends and electric current is allowed to pass
4. ninhydrin is sprayed
39
reacts with AAs to produce colored products, green/blue
ninhydrin
40
Western Blot Analysis
1. sampling and protein extraction
2. SDS-PAGE Gel Preparation
3. Protein Fractionation
4. Hybridization and Detection
41
amino acid carries a __ at pH
- positive charge
- negative electrode (cathode)
42
amino acid carries a __ at pH>pI and migrates where?
- negative charge
- positive electrode (anode)
43
- process for separating components of a mixture
- the mixture is dissolved in a substance called the mobile phase, which carries it through a second substance called the stationary phase
Chromatography
44
Three types of chromatography
1. ion-exchange chromatography
2. size-exclusion chromatography
3. affinity chromatography
45
technique for separating compounds based on their net charge
Ion exchange chromatography
46
separates molecules based on their size by filtration through a gel
Size exclusion chromatography (SEC)
47
separation method based on a specific binding interaction between an immobilized ligand and its binding partner
Affinity chromatography
48
- loss of organized structure of globular protein
- does not alter primary structure
denaturation
49
Examples of substances or process that cause denaturation
1. heat and organic compounds
2. acids and bases
3. heavy metal ions
4. agitation
50
break apart H bonds and distrupt hydrophobic interactions
heat and organic compounds
51
break H bonds between R groups and disrupt ionic bonds
acids and bases
52
react with S-S bonds to form solids
heavy metal ions
53
- ex. whipping
- stretches peptide chains until bonds break
agitation
54
- splits the peptide bonds to give smaller peptides and amino acids
- occurs in digestion of proteins
protein hydrolysis
55
what does the hydrolysis of peptide require when in the lab
- acid or base
- water
- heat
56
what does the hydrolysis of peptide require when in the body
enzymes
57
Some practical aspects of protein denaturation `
1. heat and UV
2. salts of heavy metal ions esp. Hg2+, Pb2+, Ag+
3. organic compounds such as soap, detergents, phenol, and aliphatic alcohol
58
heat and UV
- denaturation of egg white protein to become utilizable
- sterilzation using UV and heat by steam to coagulate bacterial protein
59
salts of heavy metal ions esp. Hg2+, Pb2+, Ag+
- antiseptics in low concentrations
- poisons at higher concentrations
60
any agent that produces nausea and vomiting
emetic
61
oragnic compounds such as soap, detergents, phenol, and aliphatic alcohol
- hydrophobic portions interact with hydrophobic core of protein
- hydrophilic portion H-bonded to aqueous environment
- causes swelling and unfolding of protein
62
Procedure in determination of amino acid sequence
1. hydrolysis (by acid, alkali, or enzyme)
2. identification of products
3. fitting pieces together
63
Three types of hydrolysis
1. acid hydrolysis
2. alkaline hydrolysis
3. enzymatic hydrolysis
64
involves heating in presence of 6N HCl in sealed tube at 110C for 10-100 hrs. depending on nature of protein
acid hydrolysis
65
temperature and duration during acid hydrolysis
110C
10-100 hrs
(6N HCl)
66
what is completely and partially destroyed during acid hydrolysis
completely
- trp
partially
- ser
- thr
- tyr
67
heating in presence of 4N NaOH in sealed tube at 110C 10-100 hrs
alkaline hydrolysis
68
what is used in alkaline hydrolysis
4N NaOH
69
amino acids in alkaline hydrolysis
not damaged
- trp
completely destroys
- arg
- cys
- thr
- ser
70
by proteases or peptidases
enzymatic hydrolysis
71
Two types of peptidases
1. exopeptidases
2. endopeptidases
72
enzyme that cleave external peptide bonds
exopeptidases
73
Two types of exopeptidases
1. aminopeptidases
2. carboxypeptidases
74
- sequentially cleaves peptide bonds, beginning at the N-terminal end of the polypeptide
- the liberated amino acids are identified one by one
aminopeptidases
75
sequentially cleaves peptide bonds beginning at the C-terminal end of the polypeptide
carboxypeptidases
76
cleaves internal peptide bonds
endopeptidases
77
Five types of endopeptidases
1. trypsin
2. chymotrypsin
3. elastase
4. pepsin
5. thermolysin
78
cleaves peptide bonds at the carboxyl end of the two strongly basic amino acids
trypsin
79
where does trypsin cleave
C-end
- arginine
- lysine
80
cleaves peptide bonds at the carboxyl end of the three aromatic amino acids
chymotrypsin
81
where does chymotrypsin cleave
C-end
- phenylalanine
- tryptophan
- tyrosine
82
cleaves on the carboxyl side of Gly and Ala
elastase
83
where does elastase cleave
C-end
- glycine
- alanine
84
cleaves peptide bonds at the amino end of the three aromatic amino acids
pepsin
85
where does pepsin cleave
N-end
- phenylalanine
- tryptophan
- tyrosine
86
cleaves peptide bonds at the amino end of the three aromatic amino acids and amino acids with bulky nonpolar R groups
thermolysin
87
where does thermolysin cleave
N-end
- Phe, Tyr, Trp
- Leu, Ileu, Val
88
trypsin biological source
bovine pancreas
89
Trypsin cleavage points
(C)
- Lys
- Arg
90
mouse submaxillary gland
Submaxillarus protease
91
Submaxillarus protease cleavage points
(C)
Arg
92
chymotrypsin biological source
bovine pancreas
93
chymotrypsin cleavage points
(C)
- phe
- trp
- tyr
94
Staphylococcus aureus V8 protease biological source
bacterum S. aureus
95
Staphylococcus aureus V8 protease cleavage points
(C)
- asp
- glu
96
Asp-N-protease biological source
bacterium Pseudomonas fragi
97
Asp-N-protease cleavage points
(N)
- asp
- glu
98
pepsin biological source
porcine stomach
99
pepsin cleavage points
(N)
- phe
- trp
- tyr
100
endoproteinase Lys C biological source
bacterium Lysobacter enzymogenes
101
endoproteinase Lys C cleavage points
(C)
lys
102
cyanogen bromide cleavage points
(C)
met
