December 9, 1998

CH 533-01

Isolation of -Lactalbumin

Procedure:
10 mL of Nonfat Milk
Step 1: Centrifugation.

Supernatant Sediment (Discard)
Step 2: Heat induced precipitation of Caesins.

Supernatant (Whey) Sediment (Discard)
Step 3: Gel Permeation Chromatography

Purified -lactalbumin
Step 4: UV Analysis of Fractions

Step 5: UV Scanning Analysis of Certain Fractions

Step 1--Centrifugation at 16,000 x g:
The nonfat milk was centrifuged in a 50 mL round-bottomed tube for about 45
minutes. This forces lipids and other water insoluble molecules (impurities) to sediment
at the bottom of the tube. This occurs because the spinning of the centrifuge drum places
centrifugal force on the molecules that are more insoluble in the liquid. Proteins and
nucleic acids are water soluble and remain in solution as the solid particles precipitate
into the pellet.

Step 2--Heat Induced Precipitation of Caesins:
Lowering the pH to 4.5 neutralizes the amino acid charge on the normally
insoluble caesin proteins, so that they become slightly insoluble. Raising the temperature
to 40 degrees C for about 30 minutes makes the caesins even more insoluble. -
lactalbumin remains soluble at this acidity and temperature, thus allowing the caesins to
be sedimented into a pellet using another centrifugation at 16,000 x g for 30 minutes.
The supernatant is then pushed through a 0.45 m filter to clarify the whey.

Step 3--Sephadex-100 Chromatography
The -lactalbumin was further purified by gel permeation chromatography using
a column of G-100 beads. These beads contain pores that allow proteins that are smaller
than 100,000 daltons to enter into the beads pores. All proteins greater than 100 kD are
not retained by the beads and are eluted from the column with the void volume. By this
process, the gel beads serve as a stationary phase that retards molecules between 10 and
100 kD as the eluting solvent moves the molecules throug the column. Molecules
smaller than 10,000 daltons can be absorbed by the beads too readily and not be eluted at
a consistent volume of elution buffer. Therefore, molecules between 100 kD and 10 kD
are eluted from the column nearly linearly in proportion to amount of solvent (from large
to small).
0.02 M Tris Buffer (pH 7.0) was used as the eluting solvent in our purification.
After our crude protein was added to the top of the column, we began taking 2 mL
fractions until about 30 fractions were taken. By differences in molecular weight, the -
lactalbumin was separated from the other proteins of our solution.

Step 4--UV Analysis of Fractions
Aromatic amino acid residues absorb light readily at the wavelength of 280 nm.
Therefore, we irradiated each of our fractions at 280 nm and measured the absorbance of
each. The greater the absorbance corresponds to a larger amount of protein in the
fraction. By graphing the absorbance vs. fraction, the fractions containing proteins can
be easily identified as peaks on the graph.

Step 5--Scanning UV Analysis of Isolated Proteins
This identification method allows analysis by scanning the absorbance of the
protein from 230 nm to 320 nm in 1 nm increments. Most of the functional groups of
proteins absorb light at specific wavelengths, which produces a characteristic absorption
spectrum over this range. By comparing the spectra of our isolated proteins with the
standard spectrum for -lactalbumin, the peak containing -lactalbumin could be
determined.












Data:
For Figure 1: Absorbance vs. Fraction # (First fraction was 12.5 mL, which was not
measured in the Beckman Spectrophotometer):
Fraction Number
(2 mL fractions)
Absorbance at 280 nm
2 0.0971
3 1.7110
4 2.8507
5 1.9924
6 1.1785
7 0.6470
8 0.3536
9 0.1924
10 0.2165
11 0.4834
12 1.2453
13 2.4006
14 2.5882
15 1.8012
16 0.8502
17 0.3828
18 0.1636
19 0.0846
20 0.0490
21 0.0410
22 0.0326
23 0.0331

Graphs:

Analysis of Results:
A & B: The whey was a colorless, somewhat-opaque liquid before centrifugation
and chromatography. The whey was more transparent as the thickness of the liquid
diminished.
Two major peaks are present on our graph of absorbance vs. fraction number, the
first of which contains slightly more protein that the second protein. The area
encompassed by the first protein is about 33.15, and the second proteins area is about
31.875.
Gel permeation elutes proteins in decreasing order of molecular weight over the
fractionation range of the gel. Proteins that are similar in molecular weight are eluted at a
similar elution volume. Therefore, the peak elution of an additional, similar weight
protein may be hidden by another protein that is present in a larger amount, unless very
small fractions are taken to ensure an accurate graph. Scanning spectroscopy is not very
likely to hide other proteins. This is because the scan of the fraction is usually compared
to a scan of the pure protein, thus any differences in absorbance are the result of other
proteins. The graph of our absorbance scan from 230 to 320 nm closely matches the
ideal scan for pure -lactalbumin. Therefore, only protein was probably present in our
sample of -lactalbumin after gel permeation chromatography. The second peak may
represent a mixture of proteins because we do not know the identity of the major protein
of this molecular weight, and a scanning spectrum for a pure sample of this protein was
not provided. Thus, a few proteins at about this molecular weight could contribute to
give our observed UV spectrum.
Since -lactalbumin is one of the smallest proteins in milk, it was originally
expected that it should be in the second peak. Our absorbance scan from 230 to 320 nm
showed us that the first peak contains -lactalbumin, because our graph of the scan
matches the graph of the pure -lactalbumin scan. Apparently, the second peak contains
a protein that has an even smaller molecular weight than 15,000 Daltons (that of -
lactalbumin).

Questions:

7. Most proteins contain the amino acids Trp, Tyr, and Phe, which all contain
aromatic rings. Aromatic rings are prone to absorbing light at this wavelength.

9. The concentration of -lactalbumin can be quantitatively determined using the
Beer-Lambert equation: A = Elc. In this equation, A = absorption at 280 nm, E = the
absorption coefficient (20.1 for pure -lactalbumin), and l = the path length (1 cm).
From this, the concentration of -lactalbumin can be calculated in units of grams per 100
mL. This relationship works only if the protein is pure. Other biomolecules that may
give false absorbances include nucleic acids. The aromatic purine and pyramidine rings
of the nitrogenous bases are likely to absorb at 280 nm, this is because most aromatic
rings do absorb at this wavelength.