Calcium-binding Proteins & Related

Molecules
Calcium ions act as second messengers to control a broad range of physiological effects.
Deregulation of intracellular calcium levels can result in irreversible injury and has been
implicated in several disease conditions. Thus, calcium homeostasis, the control of
intracellular Ca2+ concentration, is very tightly regulated. Calcium functions as an effector
signaling molecule by modulating the function of intracellular enzymes and voltage-gated ion
channels. Calcium-binding proteins are critical for the second messenger effects of
intracellular calcium. For example, the S100 (soluble in 100% saturated ammonium sulfate)
family of vertebrate EF-hand Ca2+-binding proteins influence diverse biological processes
including cell cycle progression, cell growth, cell motility, transcription and cell
differentiation. Calsyntenin is expressed in the ER/Golgi and on the plasma membranes of
almost all neurons. It is a calcium-binding protein that may regulate postsynaptic signaling
and APP cleavage. In presynaptic vesicles, Synaptotagmin, a highly conserved calcium-
binding protein, facilitates neurotransmitter release by triggering exocytosis.

S100A8: Products
S100A8 (also MRP8 and calgranulin A) is a 10 kDa member of the S100 family, EF-hand
superfamily of Ca-binding proteins. It is produced by neutrophils and monocytes, and forms
Ca2+-dependent heterodimer/heterotetramer complexes (termed calprotectin) with S100A9. It
functions both intracellularly and extracellularly, where it binds to RAGE and CD36. Human
S100A8 is 93 amino acids (aa) in length. It contains two EF-hand motifs (aa 12 - 47 and 46 -
81) and one high-affinity Ca2+-binding site (aa 59 - 70). There may be one splice form that
shows a 15 aa substitution for the C-terminal 14 amino acids. Although mouse S100A8 is
cleaved by MMP-2 after Asn21, it is unclear if human S100A8 is susceptible. Full-length
human S100A8 is 57% and 74% aa identical to mouse and canine S100A8, respectively.

S100A9: Products
Human S100A9 (also MRP-14, Calgranulin-B, and p14) is a 14 kDa member of the S100
family of EF-hand calcium-binding proteins. It is 114 amino acids (aa) in length and contains
short sequential modules. There is an N-terminal helical region, followed by a calcium-
binding EF-hand domain, two more helical regions, a second EF-hand domain, and three
additional helical regions. S100A9 will noncovalently heterodimerize with S100A8. In the
presence of calcium, this heterodimer will form a heterotetramer. S100A9 is expressed in
granulocytes, monocytes, and macrophages during acute and chronic inflammation. Human
S100A9 shares 62% and 57% aa identity with rat and mouse S100A9, respectively.
Ammonium sulfate precipitation

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Ammonium sulfate precipitation is a method used to purify proteins by altering their

solubility. It is a specific case of a more general technique known as salting out.

Ammonium sulfate is commonly used as its solubility is so high that salt solutions with high
ionic strength are allowed.

The solubility of proteins varies according to the ionic strength of the solution, and hence
according to the salt concentration. Two distinct effects are observed: at low salt
concentrations, the solubility of the protein increases with increasing salt concentration (i.e.
increasing ionic strength), an effect termed salting in. As the salt concentration (ionic
strength) is increased further, the solubility of the protein begins to decrease. At sufficiently
high ionic strength, the protein will be almost completely precipitated from the solution
(salting out).

Since proteins differ markedly in their solubilities at high ionic strength, salting-out is a very
useful procedure to assist in the purification of a given protein. The commonly used salt is
ammonium sulfate, as it is very water soluble, forms two ions high in the Hofmeister series,
and has no adverse effects upon enzyme activity. It is generally used as a saturated aqueous
solution which is diluted to the required concentration, expressed as a percentage
concentration of the saturated solution (a 100% solution).

In the preliminary test, the ammonium sulfate concentration is increased stepwise, and the
precipitated protein is recovered at each stage. This is usually done by adding solid
ammonium sulfate, but calculating how much ammonium sulfate to add to a solution at one
concentration to achieve a desired higher concentration is tricky, since addition of ammonium
sulfate significantly increases the volume of the solution. The amount to add can be
determined either from published nomograms or by using an online calculator. [1] Each
protein precipitate is dissolved individually in fresh buffer and assayed for total protein
content and amount of desired protein. The aim is to find the ammonium sulfate
concentration which will precipitate the maximum proportion of undesired protein, whilst
leaving most of the desired protein still in solution or vice versa.

The precipitated protein is then removed by centrifugation and then the ammonium sulfate
concentration is increased to a value that will precipitate most of the protein of interest whilst
leaving the maximum amount of protein contaminants still in solution. The precipitated
protein of interest is recovered by centrifugation and dissolved in fresh buffer for the next
stage of purification.

This technique is useful to quickly remove large amounts of contaminant proteins, as a first
step in many purification schemes. It is also often employed during the later stages of
purification to concentrate protein from dilute solution following procedures such as gel
filtration.

References

1. ^ "Ammonium Sulfate Calculator". EnCor Biotechnology Inc.

Retrieved April 2013.

Sunday, March 10, 2013

A protein purification classic: Ammonium Sulfate Precipitation An
underutilized way to purify proteins

In the 'old days' - i.e. when protein purification meant going to the
slaughterhouse to get raw materials such as placenta, brain or blood - the first
real purification step was typically an ammonium sulfate precipitation. Of course,
the natural tissue needs to be homogenized and solids removed by filtration or
centrifugation. This first clarified feedstock contains a multitude of protein
solutions, not that different from a modern-day E.coli lysate. Such lysates can be
fractionated according to the proteins' tendency to precipitate with increasing
ionic strength. Turns out that Ammonium Sulfate is a particularly suitable
precipitation reagent for this purpose. In most cases, adding a portion of a
saturated Ammonium Sulfate solution or solid Ammonium Sulfate Salt turns the
solution cloudy, indicating the precipitation of certain protein fractions. In most
cases protein purification researchers try to avoid their protein solutions turning
cloudy. In this case however, precipitation is the goal. In fact, after spinning
down the precipitate, the Ammonium Sulfate concentration can be further
increased, resulting in additional precipitation that can be removed by
centrifugation. This procedure can be repeated several times, yielding a number
of Ammonium Sulfate fractions. At this point samples are taken (precipitate and
clarified solutions from every precipitation step) and are analyzed. Since the
tendency of different proteins in the lysate differs as a function of Ammonium
Sulfate fractions, different proteins are separated in distinct fractions. One ends
up with a handful of fractions and their corresponding protein content.

How do you use this data to establish a purification procedure for a

particular target protein?

In the best case scenario you select the fraction that precipitated the protein of
interest. Then you'd try to bring this precipitate back into solution, for instance
by reducing the Ammonium Sulfate concentration and adjusting the buffer
composition. Dialysis is a simple way to achieve this. Once clarified, further
purification steps can be added if necessary.

In cases where the precipitated protein does not re-solubilize, the 'earlier'
fraction should be chosen, i.e. the lower Ammonium Sulfate concentration
fraction that contains the protein in a non-precipitated form. This fraction
contains more contaminants, but has fewer contaminating proteins than the
starting material.

How do you use the protein sample composition of Ammonium sulfate fractions to establish a purification
procedure for a particular target protein?

Practical tips for Ammonium Sulfate precipitation experiments:

Carry out the precipitation reaction on ice (or thermostatted at a specific temperature),
in a glass beaker with a magnet bar, on a stir plate
Sequential operation: for each fractionation step let the precipitation reaction come to
completion by incubating for a minimum of 10 min. This will procedure may yield
incomplete precipitation, but is much faster
Parallel operation: prepare multiple precipitation experiments, adding a final
Ammonium Sulfate concentration of 10%, 20%, , 30% etc. Over night incubation assures
that the precipitation has come to completion.
Working with solid Ammonium Sulfate allows to explore high Ammonium Sulfate
concentrations:
- Dry Ammonium Sulfate by baking overnight at 120C and grinding to a fine powder
(wear a dusk mask!)
- Add the Ammonium Sulfate powder very slowly. Don't add entire chunks, avoid
clumping in the stirred solution. Sprinkle in the powder and wait for it to dissolve
completely.
- Add pre-weighted amounts of ammonium sulfate.
Working with Ammonium Sulfate is more convenient. Just prepare a saturated
Ammonium Sulfate solution by adding 750 g of Ammonium Sulfate to 1 liter of destilled
water and stir for 15 minutes. The supernatant of this solution is 100% saturated.
Local Ammonium Sulfate concentration spikes can be avoided by working with a
solution. However, a saturated solution dilutes the lysate, increases working volume and
limits the Ammonium Sulfate concentration to the low Ammonium Sulfate concentration
regime. Regardless, add the Ammonium Sulfate solution slowly and stir vigorously.
Avoid foam formation or frothing (this can oxidize the protein)
Take samples of the solution and the precipitate for analysis of protein composition
Dilute the precipitate with buffer to the original lysate volume to allow for better
comparison of yields between fractionation steps

Useful online tools for Ammonium Sulfate precipitations:

Online calculator to calculate the amount of solid Ammonium Sulfate required to add to a
specific volume of a solution to get a specific percentage saturation at a specific
temperature;

Ammonium Sulfate precipitation demonstration experiment for the isolation of

Hemoglobin, Amylase and Protease by ammonium sulfate precipitation

Ammonium Sulfate precipitation at work: recent examples of the

utility of this economic protein purification method.
Mahn A, & Ismail M (2011). Depletion of highly abundant proteins in blood plasma by
ammonium sulfate precipitation for 2D-PAGE analysis. Journal of chromatography. B,
Analytical technologies in the biomedical and life sciences, 879 (30), 3645-8 PMID:
21963274

Moore PA, & Kery V (2009). High-throughput protein concentration and buffer exchange:
comparison of ultrafiltration and ammonium sulfate precipitation. Methods in molecular
biology (Clifton, N.J.), 498, 309-14 PMID: 18988034

Park JW, Lee SG, Song JY, Joo JS, Chung MJ, Kim SC, Youn HS, Kang HL, Baik SC, Lee WK,
Cho MJ, & Rhee KH (2008). Proteomic analysis of Helicobacter pylori cellular proteins
fractionated by ammonium sulfate precipitation. Electrophoresis, 29 (13), 2891-903
PMID: 18546177

Ko KY, & Ahn DU (2007). Preparation of immunoglobulin Y from egg yolk using
ammonium sulfate precipitation and ion exchange chromatography. Poultry science, 86
(2), 400-7 PMID: 17234857