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0.3 M urea
0.3 M glucose
0.3 M ethylene glycol
0.3 M glycerol
2. Procedure
Add 2 drops of suspension in each
solution
Record hemolysis time
Threshold: 10 minutes
Permeability and Partition Coefficients
1. Solutions
RBC suspension
o 7 drops blood
o 5 mL 0.9% NaCl
(+) control
o 5 drops RBC suspension
o 5 mL dH2O
(-) control
o 5 drops RBC suspension
o 5 mL 0.2M NaCl
0.6 M methanol
0.6 M ethanol
0.6 M isopropanol
0.6 M n-butanol
2. Procedure
Immerse solutions in an ice bath are
take note of temperature
Add 2 drops of suspension in each
solution
Record hemolysis time
Threshold: 10 minutes
Effect of Polar Groups on Permeability
1. Solutions
RBC suspension
o 7 drops blood
o 5 mL 0.9% NaCl
(+) control
o 5 drops RBC suspension
o 5 mL dH2O
(-) control
o 5 drops RBC suspension
o 5 mL 0.2M NaCl
0.3 M chloroform
0.3 M hexane
0.3 M ethylene glycol
0.3 M isopropanol
2. Procedure
Add 2 drops of suspension in each
solution
Record hemolysis time
Threshold: 10 minutes
ANIMAL PHYSIOLOGY: FIRST LAB EXAM / A.B.C.
B. DISCUSSION
The Cell Membrane
Phospholipid bilayer
Outer: hydrophilic heads, inner: hydrophobic
tails
Other components: membrane proteins,
other lipids (i.e. cholesterol)
Selectively permeable
Regulates the passage of materials into and
out of the cell
o Nutrition
o Maintenance of irritability (i.e.
sensitivity to stimuli, fluidity of
membrane)
o Homeostasis (milieu intrieur)
Transport Through the Membrane
Passive
o Simple diffusion the net movement
of solutes through a selectively
permeable membrane from an area
of higher energy to an area of lower
energy
(higher
to
lower
concentration) by random thermal
motion
o Facilitated diffusion assisted by
proteins embedded in the membrane
but still across a concentration
gradient
Channel proteins provide a
path for lipid insoluble
molecules to pass through
Carrier proteins undergo
conformational changes to
transport solutes
Active
o Occurs against the concentration
gradient and, therefore, requires
energy
o This energy is usually provided in
the form of adenosine triphosphate
(ATP)
Osmosis
o Diffusion of water through a
selectively permeable membrane
o Water moves according to its own
concentration gradient
Osmoticity vs. Tonicity
Osmoticity refers to the relationship of
solutions on opposite sides of an ideal
selectively permeable membrane (one that
2
Degree of Dissociation
Solute
Chloroform
Hexane
Ethylene Glycol
Isopropanol
Molecular Weight
62.07 g/mol
66.06 g/mol
92.09 g/mol
180.00 g/mol
Dielectric Constant
4.81
2.0
37.0
17.9
Partition Coefficient
(k)
0.14
0.26
0.64
7.7
A
partition
coefficient
measures
hydrophobicity in that it is the ratio of a
molecules solubility in a lipid and the same
molecules solubility in water
Overtons
Rule
states
that
higher
hydrophobicity
relates
to
higher
permeability; there is a positive relationship
between the partition coefficient and time of
hemolysis
Non-polar compounds tend to have higher
solubility in water and (way) lower solubility
in water and, therefore, higher partition
coefficients; molecules with longer carbon
chains permeate the membrane more easily
than molecules with shorter carbon chains
Because of the hydrophobic nature of a bulk
of the cell membrane, substances with
ANIMAL PHYSIOLOGY: FIRST LAB EXAM / A.B.C.
Effect of pH
McIlvaines Solutions
pH
3
4
5
7
8
0.2 M Na2HPO4
(mL)
4.11
7.71
10.30
16.47
19.45
0.1 M citric
acid (mL)
15.89
12.29
9.70
3.53
0.55
Effect of Temperature
B. DISCUSSION
Enzymes
Regulation by Inhibition:
o Competitive inhibitor binds to active
site
o Non-competitive inhibitor binds to
enzyme at site other than active site
Salivary Amylase
Also known as ptyalin
Hydrolyzes starch and glycogen through its
action on -1,4- glycosidic bonds
Starch dextrins maltose
Optimum pH range: 5.6 to 6.9
Optimum temperature: 38
Starch-Iodine Test
Starch is a polymer of -D-glucose
molecules bound by -linkages; it forms a
helical structure
This structure is significant because it forms
a complex with iodine molecules as the I2
particles nestle themselves within the helix
and for a blue-black complex
In the lab, this can be used to test the
presence of starch in a certain solution
Achromic Point
The point at which no blue-black color is
produced when a drop of digestive mixture
is added to I2-KI
The non-formation of blue, black, or violet is
indicative of the cleavage of the starch in
Enzyme Kinetics
Effect of Enzyme Concentration
o There is a positive relationship
between enzyme concentration and
the rate of reaction or enzyme
activity
o Graph does plateau, however, since
substrate concentration is kept
constant
Effect of Substrate Concentration
o Theoretically, there should also be a
positive
relationship
between
substrate concentration and the rate
of reaction or enzyme activity
o However, the reality is that enzymes
occur in finite quantities in the
bodyeven if the substrate is
abundant,
enzyme-substrate
complexes can only form if there are
available enzymes
Michaelis-Menten Equation
Reaction
Velocity
vs.
Substrate
concentration
The Michaelis-Menten constant (Km)
describes the affinity of the enzyme and
substrate; Km = affinity
At Km, the rate of reaction is at 50% of Vmax
Lineweaver-Burke Equation
1/Reaction
Velocity
vs.
1/Substrate
Concentration
To linearize the M-M plot
Slope of Line Equation = Km / Vmax
x-intercept of line = - 1 / Km
y-intercept of line = 1 / Vmax
Thigmotaxis
Observe Paramecium slide prepared using
cotton fiber method; note difference in
movement when anterior and posterior ends
hit thread
Chemotaxis
1. Solutions
0.2% HCl
0.2% CH3COOH
0.1 M NaHCO3
3% NaCl
5% sucrose
2. Procedure
Prepare Paramecium slides without
cotton fibers and agar
Dip cotton thread into each of the
solutions listed above and lay it on the
slide
Observe reaction of organism
Geotaxis & Phototaxis
Geotaxis
o Test tube was filled of the way up
with Paramecium culture then set
upright in a test tube rack
o Positioned under a bright light for 15
minutes
Phototaxis
o Test tube was filled of the way
with Paramecium culture then
sealed with a rubber stopper
o Tube was laid on its side and its
upper surface was covered with foil
o Light was shone from below the tube
Galvanotaxis
Ringers Solution (1 L)
6.5 g NaCl
0.14 g KCl
0.12 g CaCl2
0.20 g NaHCO3
0.01 g Na2HPO4
4 x 0.5 x 0.3 cm Paraffin well on slide
Fill well with 3-5 drops of Ringers solution
and culture
Attach uninsulated ends of 24-gauge wires
with tape; wires connected to batteries to
close the circuit
Voltage was adjusted to 4.5, 6, 7.5, 9.0, and
12 V using multiple batteries
ANIMAL PHYSIOLOGY: FIRST LAB EXAM / A.B.C.
o
B. DISCUSSION
Paramecium
Kingdom Protista, Phylum Ciliophora;
eukaryotic, unicellular
animal-like = locomotory, no cell wall,
heterotrophic
easily excitable cell membrane
Gross Morphology
o
o
Movement
Locomotion
primarily
controlled
by
metasynchronous ciliary beating (effective
stroke propels forward, recovery stroke
keeps cilia flexed)
Spiral motion that appears zig-zag, rotates
(counterclockwise) along longitudinal axis
due to oblique ciliary beating
Digestion
Distilled water
Paramecium culture
M/500 KCN
2.5% NaCl
3% NaCl
5.0% NaCl
7.5% NaCl
10.0% NaCl
Contractions per
Minute
20
15
6
8
8
7
7
2
Thigmotaxis
Negative response to stimuli
ANIMAL PHYSIOLOGY: FIRST LAB EXAM / A.B.C.
Difference
between
reactions
when
opposite ends are touched is due to type of
channels located on each
Anterior end: reverse movement
o Depolarization of membrane by
influx of Ca2+, cilia slow down and
Paramecium reverses
Posterior end: faster forward swimming
o Hyperpolarization of membrane by
efflux of K+, cilia accelerate
Chemotaxis
Defined as the directional response to
chemical stimuli (innate mechanism)
Not to be confused with chemotactic
response, which refers to the reactions of
Paramecium to a new or unknown stimuli
Chemokinesis, on the other hand, is random
change in movement of organism due to
chemical stimuli
Poositive chemotactic response to weak
acids and sugar solutions (acetic acid and
sucrose)
o Paramecium thrive in slightly acidic
environments
as
the
main
component of their diet, bacteria,
regularly occur in slightly acidic
media
o Causes the hyperpolarization of the
membrane
(more
negative
membrane potential) and, thus,
acceleration of ciliary beat
Negative chemotactic response to strong
acids, bases, and salt solutions (HCl,
NaHCO3, and NaCl)
o Salt solutions are hyperosmotic and,
thus, cause the depolarization of the
membrane as water rushes out of
the cell (also, Paramecium is a
freshwater resident)
o Strong acids have adverse effects
on Paramecium; high concentration
of H+ causes depolarization of
membrane
o Bases cause the depolarization of
the membrane due to the low
concentration of H+
Geotaxis
Response to gravity
Paramecium exhibit negative geotaxis
Individual organisms gather near the surface
of the medium
1
0
Metabolic Rate
Basal Metabolic Rate (MR in g-cal/hr)
Volume of CO2
4. Graphs needed:
O2 concentration vs. CO2 concentration
Mass-specific metabolic rate vs. mass
Respiration rate vs. mass
B. DISCUSSION
Metabolism
Sum of all biochemical activity occurring
within an organism
Related to the amount of energy that the
organism needs to function
Two types:
o Anabolism reductive reaction to
synthesize bigger molecules using
smaller ones; requires energy
o Catabolism oxidative breakdown of
complex molecules into smaller
ones; releases energy
Two pathways based on the presence of O2:
o Aerobic in the presence of oxygen;
36-38 molecules of ATP
o Anaerobic in the absence of
oxygen; 2 molecules of ATP
Measurement of Metabolic Rate
Metabolic reactions transform chemical
energy into heat energy; measure the
amount of heat energy transformed per unit
time measures metabolic rate
Measurement Methods:
o Calorimetry measuring heat output
Direct measuring heat
produced directly (i.e. ice
bath calorimeter)
Indirect based on gas
exchange; assumes that the
intake of O2 and the output of
CO2
corresponds
to
metabolic activity and relates
the concentrations of these
to heat production through
stoichiometry
o Radioisotope Labeling
o Respirometry
Warburg Method
1
1
Pressure
measurement
of
gases
Gilson Respirometer
Absorption of CO2
animal releases
1
3