Enzyme Assay: Lactase

Enzyme Assay: Lactase

[See Hartman, Suskind & Wright, Principles of Genetics Lab Manual, (1965). pp. 52-58.)]

hydrolysis_of_lactose

Lactose, (milk sugar) is a disaccharide formed from galactose and glucose. Its beta-galactosidic bond must be hydrolyzed to yield its component monosaccharides before they can be absorbed by the body. In humans, the enzyme lactase performs this task. Many individuals lose the ability produce lactase, and therefore “lactose intolarent” after they enter their teens. As a result, they suffer GI upset when they consume milk products (gas, abdominal pain, diarrhea). Lactase tablets may reduce this problem. We will be assaying the lactase content in commercial tablets, and use this enzyme as a typical enzyme in our subsequent studies.

ONPG_hydrolysis

The substrate used in the assay of this enzyme is o-nitrophenyl–D galactoside (ONPG), which, upon hydrolysis of the -galactosidic bond, yields galactose and o-nitrophenol, a yellow compound (absorption max = 450 nm) ( CRC Handbook: #p679, Merck Index, #6541).

Enzyme activity is proportional to the increase in A 450 during incubation.
Lactase_assay/ONPG hydrolysis

As in many enzyme assays, adjustments in concentrations and volumes may be needed for optimum results. Keep careful track of how you set up your experiment. This is often best accomplished by diagramming the procedure .

Materials and equipment: (per team of two students)

SUPPLIES: PER CLASS:
fresh lactase (suspend in buffer) (9000 unit tablet suspended in 90 mL buffer)
20 mM o-nitrophenyl–D galactoside (20 mL ONPG)
0.1 M PO 4 buffer, pH 5.5 (60 mL)
0.01 M PO 4 buffer, pH 5.5 (120 mL)
4% K 2CO3 (100 mL)

Distribute to each table:
20 mL dH2O in small flask
7 mL reaction mix* in 13 x 100 mm tube
1 mL 1:200 diluted enzyme (0.5 units/mL) in 13X 100 mm tubes
7 mL 4% K 2CO3 in 13 x 100 mm tube

EQUIPMENT
25 mL grad cylinder
100 mL grad cylinder
test tubes: five 13×100 in rack
200 and 1000 micropipets and tips
vortex
Eppendorf Repipeter with 10 mL syringe
37 C hot block, 13 mm holes
stopwatch
spectrophotometer
2 cuvettes in rack at spectrophotometer

01_suspend_lactase_Pa172076md
Suspending lactase in buffer

1. a: Record the brand of lactase, labeled number of units of lactase/tablet and the expiration date.
b: Weigh one lactase tablet, note whether 9,000 FCC or 3,000 FCC units/tablet.
c: Grind in a mortar and pestle until finely ground.
d: Suspend/dissolve to 100 units/mL: Grind a tablet in about 5 mL of chilled 0.01 M PO4 buffer, pH 7.
For 9,000 unit tabs, q.s. to 90 mL with same buffer, including mortar and pestle rinses.
For 3,000 unit tab, q.s. to 30 mL (Solution will be cloudy because of undissolved binder.)
e: Dilute 1:200: Add 0.1 mL of enzyme suspension into 19.9 mL 0.01 M PO4, pH 5.5, in a 25 mL grad cylinder.
Most enzymes should be kept on ice until ready to use, this may not be necessary for lactase.

*2. Prepare reaction mix (Rxn Mix): Per desk: Per class of 20:
0.1 M PO4 pH 5.5 buffer 5.6 mL 56 mL
20 mM ONPG 1.4 mL 14 mL

3. a. Copy the following table into your notebook.
b. Then set up a series of numbered 13×100 mm test tubes as follows.
c. Add water to the tubes first, then RxnMix with the repeater pipetter. (Not the enzyme yet

09.5_prewarm_tubes_Pa172074

4. Vortex and pre-warm these tubes in a 37 C hot block for two minutes.

5. At 30 second intervals, add listed uL of enzyme, vortex, start a stopwatch with 1st tube, place in 37 C hot block.

10_finished_assay_close_Pa172073md
Lactase assay, after incubation

6. After exactly 15 minutes, add 1.0 mL 4% K2 CO3 down the side of the first tube, mix and remove from hot block. At 30 second intervals, repeat 4% K2 CO3 addition for each of the successive tubes, mix and set aside.

7. Read the absorbency at 450 nm, record in your notebook table, graph and discuss results.

8. Calculate the number of units of lactase (1.000 OD unit/15 min) in the original tablet. (See following protocol). Compare with other brands of lactase.
CALCULATION OF LACTASE ACTIVITY/TABLET:
If 1 unit of lactase produces an OD of 1.000/15 min., and the assay was run for 15 mins:
units/tablet = A 450 x 100 mL/tablet x dilution factor x 1/(aliquot in mL

REAGENTS, MATERIALS AND CALCULATIONS FOR LACTASE ASSAY page 26

19 September 1993. rvsd 25 October 1994, 18 Sept 95, 20 Sept. ’96
0.1 M PO 4 pH 7.0 BUFFER:
For 200 mL, weigh out: 1.0 g KH2PO 4
1.8 g Na 2HPO4
dissolve in 200 mL H 2O, check pH, adjust to 7.0 if nec. with either H 3PO4 or NaOH. Store at 4C.

0.01 M PO 4 pH 7.0 BUFFER : (for suspension and dilution of enzyme)
Q.s. 50 mL of pH 7.0 0.1 M PO 4 buffer to 500 mL with dH 2O.

20 mM o-nitrophenyl–D galactoside (ONPG): (chromogenic substrate)
Weigh out: 602 mg ONPG
dissolve in about 80 mL 0.01 M PO4 buffer, pH 7.0 with swirling and slight warming.  q.s. with buffer to 100.0 mL.]
REAGENT TO HALT REACTION:
4% K 2CO3 : dissolve 8 g K 2CO3 in 200 mL dH2 O, stir to dissolve.
MATERIALS AND EQUIPMENT for team of four assaying given brand of lactase:
(two sub teams each perform an assay) 10/25/94, rvsd 18 Sept ’95, 20 Sept. ’96

EQUIPMENT:
mortar and pestle
100 mL graduated cylinder
ice bath
5.0 mL pipet (for dH 2O)
pipet bulb or helper
2 x 200 lambda micropipettes
(for ONPG and enzyme)
2 x 1000 lambda micropipettes
(for buffer and 4% K2CO3 )
2 16 x 150 mm test tubes
10 13 x 100 mm tubes
two test tube racks for 13×100 tubes
37C hot block for 13 x 100 mL
2 stopwatches
spectrophotometer, warmed up
at spectrophotometer:
cuvettes in rack
wipettes
SUPPLIES:
lactase tablets
100 mL 0.01 M PO 4 buffer
(to suspend and dilute enzyme)
30 mL dH 2O in 125 mL flask
(to make up assay set)
3 mL 20 mM o-nitrophenyl–D galactoside
(ONPG)
15 mL 0.1 M PO 4 buffer, pH 7.0
(for assay tubes)
15 mL 4% K 2CO

Advertisements

Cells: The Functional Units of Organisms

Cells: The Functional Units of Organisms

Robert Hooke first described cells in cork in Micrographia, pp 112-116 (1665). Curator of Instruments for Royal Society, viewed thin slices of cork under 30x lens, showed network of tiny compartments, he called cellulae (little rooms)

Later, the unified cell theory developed from the combined works of a botanist, Mattias Schleiden who published a study of the cellular nature of plant tissues in 1838, and a zoologist, Theodor Schwann, who studied animal tissues and illustrated cells he found in cartilage:

Robert Brown 1833, English botanist, using improved lenses, noted that every orchid epidermis cell had round structure he termed nucleus

Matthias Schleiden 1838, German botanist, concluded all plant tissues composed of cells, and embryonic plant arose from a single cell.

Theodor Schwann 1839, German zoologist, examined cartilage tissue (better cellular definition in cartilage), concluded all animal tissues are composed of cells

Rudolf Virchow 1855, Ger Physiol, concluded cells arose only by division of preexisting cells: Omnis cellula e cellula.

The unified cell theory, developed from their work:
UNIFIED CELL THEORY (three tenets):
1) all organisms made of cells
2) cell is the basic unit of structure
3) cells arise only from preexisting cells.

We will repeat some of these classical studies by preparing and examining a variety of plant and animal tissues to search for evidence of their cellular composition. Remember to strictly follow correct protocol for microscope use.

MATERIALS:
(Wash all tools immediately after each use. Do not let material dry out on them.)
single edge sharp razor blades
dropper bottle of dH 2 O
0.3% methylene blue stain
clean slides and cover slips
forceps and fine scissors
cork
fresh onion
cartilage from breast bone of a young chicken
Prepared slide:
hyaline cartilage, trachea, H&E, H 680 

1. CORK: Repeat the experiment of Robert Hooke: First slice off a “good clear piece of cork” with a single-edged razor blade to produce a smooth, clean surface. Then shave a wedge of cork as thin as possible from the clean surface. Examine the thinnest edge of the slice under the microscope, and illustrate its structure seen at 400x. What exactly are you seeing?

2. ONION EPITHELIUM: Prepare a slide with three spots: 1) dry, 2) drop of water, 3) drop of methylene blue. Slice a wedge from a fresh onion and discard the outer layer or two. Separate a fresh layer and break it back to the membranous outer layer of epithelium. Peel off epithelium with forceps, cut into three small pieces, apply one to each of the three prepared spots on the slide (keep flat). Cover with cover slips. Write out your observations of each: 1) dry mount, 2) wet mount with water, 3) wet mount with methylene blue. Which mounting technique resolved the most detail? Illustrate any evidence of stained cells seen at 100x and 400x (on the same page). Label: plasma membrane, nucleus, nucleolus, and cytoplasm. Search for stomata, especially in outer epithelium. Here is a stoma stained.

Onion Epithelium stained. Note the nuclei apparent in many of the cells.

3. CHICKEN CARTILAGE: Slice an ultra-thin section of the hyaline cartilage from a chicken breast bone as you did for the cork in exercise 1. Prepare a wet mount in water and a wet mount in methylene blue. Compare the two views. Illustrate any evidence of stained cells seen at 400x. Why was this material a fortunate choice for Schwann to first study animal cells? (cells = chondrocytes. The space between = matrix). (Wash tools immed.)

Chicken Cartilage stained. Chondrocytes are embedded in a matrix which does not take the stain as well as the cells.

Hyaline_Cartilage_400x_PA112028lbd

HYALINE CARTILAGE, PREPARED SLIDE: Examine hyaline cartilage as seen in the prepared slide (H 680) from a cross section of the trachea. Compare with the section you prepared in 3, illustrate at 400x. Label: matrix, lacunae and chondrocytes.

5. CHICKEN MUSCLE: For a contrasting tissue, cut a very thin section of muscle, suspend in stain, squash under a cover slip. Note muscle fibers at 100x, striations at 400x. The greenish oblong features running along and above the muscle fiber are mitochondria. Here is a prepared slide of skeletal muscle labeled.

 

Lactase Assay: Reagents

Lactase Assay: Reagents

[See Hartman, Suskind & Wright, Principles of Genetics Lab Manual, (1965). pp. 52-58.)]Lactase Assay Reagents

The substrate used in the assay of this enzyme is  o-nitrophenyl–D galactoside (ONPG), which, upon hydrolysis of the -galactosidic bond, yields galactose and  o-nitrophenol, a yellow compound (absorption max = 450 nm) ( CRC Handbook: #p679,  Merck Index, #6541). Enzyme activity is proportional to the increase in A450 during incubation.

REAGENTS, MATERIALS AND CALCULATIONS FOR LACTASE ASSAY page 26

0.1 M PO 4 pH 7.0 BUFFER:
For 200 mL, weigh out: 1.0 g KH2PO 4
1.8 g Na 2HPO4
dissolve in 200 mL H 2O, check pH, adjust to 7.0 if nec. with either H 3PO4 or NaOH. Store at 4C.

0.01 M PO 4 pH 7.0 BUFFER : (for suspension and dilution of enzyme)
Q.s. 50 mL of pH 7.0 0.1 M PO 4 buffer to 500 mL with dH 2O.

20 mM o-nitrophenyl–D galactoside (ONPG): (chromogenic substrate)
Weigh out: 602 mg ONPG
dissolve in about 80 mL 0.01 M PO4 buffer, pH 7.0 with swirling and slight warming. 
q.s
. with buffer to 100.0 mL.

This reagent is unstable over periods of weeks at 4 C.  Aliquots may be frozen to stabllize during storage.

REAGENT TO HALT REACTION:
4% K 2CO3 : dissolve 8 g K 2CO3 in 200 mL dH2 O, stir to dissolve.

MATERIALS AND EQUIPMENT for team of four assaying given brand of lactase:
(two sub teams each perform an assay) 10/25/94, rvsd 18 Sept ’95, 20 Sept. ’96

EQUIPMENT:
mortar and pestle
100 mL graduated cylinder
ice bath
5.0 mL pipet (for dH 2O)
pipet bulb or helper
2 x 200 lambda micropipettes
(for ONPG and enzyme)
2 x 1000 lambda micropipettes
(for buffer and 4% K 2CO3 )
2 16 x 150 mm test tubes
10 13 x 100 mm tubes
two test tube racks for 13×100 tubes
37C hot block for 13 x 100 mL
2 stopwatches
spectrophotometer, warmed up
at spectrophotometer:
cuvettes in rack
wipettes
SUPPLIES:
lactase tablets
100 mL 0.01 M PO 4 buffer
(to suspend and dilute enzyme)
30 mL dH 2O in 125 mL flask
(to make up assay set)
3 mL 20 mM o-nitrophenyl–D galactoside
(ONPG)
15 mL 0.1 M PO 4 buffer, pH 7.0
(for assay tubes)
15 mL 4% K 2CO3
CALCULATION OF LACTASE ACTIVITY/TABLET:

If 1 unit of lactase produces an OD of 1.000/15 min., and the assay was run for 15 mins:

units/tablet = A 450 x 100 mL/tablet x dilution factor x 1/(aliquot in mL)

Features of Selected Bones

 

Features of Selected Bones

Illustrate these disarticulated bones and include the listed features with labels (handle with care and respect please, they are real & fragile).
Enter in your book on pages in this order:

First set of pages:
Facing pages (illustrations 1 and 2):
1) cervical and thoracic vertebra
2) lumbar vertebra and articulated atlas and axis
3) sacrum
4) scapula

Second set of pages
Facing pages (illustrations 5 and 6):
5) humerus front and back
6) radius and ulna
7) hand and wrist
8) os coxa
9) ankle and foot

Articulated C-1 and C-2 :labeled drawing of vertebra( top rear view of the articulated bones) (one picture)
atlas: no centrum, articular surface for odontoid process, no spinous process
axis: odontoid process (dens) (Martini’s 5th: p 215)
Here is a labeled view of articulated C-1 and C-2.

Here is a labeled view of atlas and axis with spinous process at top.


 


Cervical : most delicate of the vertebrae (Martini’s 5h: p 215-218)
Unique cervical features: transverse foramina, bifurcated spinous process


Thoracic : label features common to all vertebrae:
centrum, neural arch, vertebral foramen, pedicle, spinous process, lamina, superior articular processes, transverse process, Draw small side view of a thoracic vertebra showing the demifacets and the vertebral notches which form intervertebral foramina for spinal nerves. Unique thoracic features:demifacets for articulation with rib head, seen from side (except for 11th and 12th.), articular facets on transverse processes (for rib tubercle) and long delicate spinous processes

Here is a labeled view of a thoracic vertebra.
Here is a labeled side view of articulated thoracic vertebrae.
Here is a labeled picture of the articulation of ribs with vetebra.


Lumbar : heavy centra, broad heavy spinous process, transverse process lacks facets


Sacrum:(posterior view): dorsal sacral foramina, superior articular facet, auricular surface (on sides, for os coxa), (ala), median sacral crest, sacral canal, sacral hiatus.
Here is a labeled view of the sacrum.



Scapula : (posterior view): labeled drawing scapulaacromion process, coracoid process, spine of the scapula, supraspinous fossa, infraspinous fossa, glenoid fossa, vertebral border, axillary border
Here is a labeled image of the posterior scapula.


23.4. Bones of the arm: (two pages: two views of humerus on first,
ulna and radius on facing page)

humerus_ant&post_PB012145-46
Humerus: (anterior and posterior views): head, greater tubercle, lesser tubercle, intertubercular groove, deltoid tuberosity, lateral epicondyle, medial epicondyle, trochlea, coronoid fossa, capitulum, olecranon fossa


 


Ulna : (lateral view) olecranon process, trochlear notch (semilunar notch), coronoid process, head, styloid process, radial notch
Radius : head, neck, radial tuberosity, styloid process, ulnar notch

Here is a labled view of the right ulna and radius.
(Here it is in vertical orientation.)
Here is a labeled view of supination and pronation.


Articulated bones of the wrist and hand, labeled drawing, wrist and handventral view (trace around your hand as a model):
include distal ulna and radius
proximal row of carpals: scaphoid lunate triquetrum pisiform
distal row of carpals: trapezium trapezoid capitate hamate
first through fifth metacarpals, proximal, middle and distal phalanges.

Here is a labeled view of the bones of the hand.
Here is another labeled view of the right forearm before and after pronation.


femur_chicken_collage_numbered_sm
Here is a collage of chicken femur pictures for you to label.



Os coxa, lateral view with acetabulum and obturator foramen: labeled drawing, os coxa (Note differences between male and female .)
a: ilium: iliac crest, anterior superior iliac spine, anterior inferior iliac spine, posterior superior iliac spine, posterior inferior iliac spine, greater sciatic notch
b: pubic bone: pubic tubercle, inferior ramus
c: ischium: ischial spine, ischial tuberosity, ischial ramus

Here is a labeled lateral view of the right os coxa.
Here is a labeled view of male versus female pelvices.
Here is a labeled view of the female pelvis.
Here is a labeled view of the male pelvis.


 


Articulated bones of the ankle to metatarsals , top view:labeled drawing, ankle and foot
talus, calcaneus, navicular, medial cuneiform, intermediate cuneiform, lateral cuneiform, cuboid, first through fifth metatarsals
Here is a labeled view of the bones of the right foot.


tarsals_superior_PB060027
Detail, superior view tarsals:


VERTEBRA (on facing pages: two pictures on each page, same scale, superior views for all)