Characterization of Whey Protein Isolate (WPI) Using Multi-Detector Size-Exclusion Chromatography

APPLICATION NOTE

Characterization of Whey Protein Isolate (WPI) using multi-detector size-exclusion chromatography

MOLECULAR WEIGHT Introduction Whey is a by-product of the cheese-making process, and Whey Protein Isolate (WPI) is MOLECULAR SIZE a dietary supplement and food ingredient created by separating components from milk. It contains a high percentage of pure protein and can be pure enough to be virtually PROTEIN AGGREGATION lactose free, carbohydrate free, fat free, and cholesterol free.

Whey protein Isolate is of high nutritional value, and has become an important source of functional ingredients in various health-promoting foods. It is widely used in infant formula to provide a natural source of amino acids for optimal growth and development, and is also often used as an emulsifier and stabilizer in the food industry. WPI is also popular among athletes because of its ability to be digested very rapidly and help return the post-workout body back from a catabolic state to an anabolic state. It has been reported that WPI may possess anti-inflammatory or anti-cancer properties.

WPI consists of four major components: β-lactoglobulin , α-lactalbumin, serum albumin, and immunoglobulins. β-lactoglobulin is the major component of cow and sheep’s WPI, and is also present in many other mammalian species. However, it is not present in human breast milk. Approximately 85% of milk-allergic children outgrow their allergy by the age of three but the remaining 15% remain allergic. Bovine β-lactoglobulin (BLG) is the major allergen in cow’s milk, so dairy product manufacturers need to prove the presence or absence of β-lactoglobulin to ensure their labelling satisfies the requirements of milk- allergy suffers. The β-lactoglobulin is a relatively small protein of 162 amino acids, with a molecular weight of 18.4 kDa, and in physiological conditions it exists predominantly as a dimer.

α-lactalbumin is a protein present in the milk of almost all mammalian species, and is involved in the production of lactose. The molecular weight is 14,178 kDa.

Serum albumin is produced by the liver, its primary function is as a carrier protein for steroids, fatty acids and thyroid hormones in the blood, and it plays a major role in stabilizing extracellular fluid volume by contributing to oncotic pressure. Serum albumin is a globular, water-soluble protein with an approximate molecular weight of 65 kDa.

Immunoglobulin (Ig), also known as an antibody, is used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. In placental

mammals there are five antibody isotypes known as IgA, IgD, IgE, IgG and IgM. A typical Immunoglobulin has a molecular weight of 150 kDa.

In this application note, a sample of WPI was characterized using the Viscotek TDAmax. The 4 main components in this mixture were separated and individually characterized.

Materials and methods The samples were analyzed on a Viscotek TDAmax system from Malvern Instruments, with refractive index (RI), ultraviolet (UV), 7°angle light scattering (LALS), 90°angle light scattering (RALS) and intrinsic viscosity (IV) detectors.

The samples were separated with two Viscotek P-columns (300 x 7.8 mm ID), 1 x P4000 and 1 x P3000, with a protein molecular weight exclusion limit of 700 kDa and 1000 kDa respectively.

The mobile phase was 0.1M pH7.0 phosphate buffer, around 500 µg of total material was injected for each measurement. The chromatograms were analyzed using the conjugate analysis calculations in the OmniSEC software.

Results and discussions Components characterization The X axis was Retention Volume (RV), Y axis was RI (refractive index), UV (ultraviolet), RALS (Right angle light scattering), LALS (low angle light scattering), DP (viscometer) respectively.

2 Characterization of Whey Protein Isolate (WPI) using multi-detector size-exclusion chromatography APPLICATION NOTE

Immunoglobulin Serum albumin β-lactoglobulin α-lactalbumin

Peak RV - (mL) 17.057 18.174 19.238 20.349

Mn - (Daltons) 150,329 70,310 30,745 15,410

Mw - (Daltons) 151,366 70,469 31,847 15,549

Mz - (Daltons) 152,485 70,635 33,125 15,697

Mp - (Daltons) 127,955 69,619 30,683 15,800

Mw / Mn 1.007 1.002 1.036 1.009

IV - (dL/g) 0.0596 0.0506 0.0339 0.0289

Rh(w) - (nm) 5.181 3.833 2.564 1.921

Wt Fr (Peak) 0.024 0.064 0.737 0.175

The dn/dc of all proteins was set to 0.185.

Peak 3, with a molecular weight of 33 kDa and 75% weight fraction, corresponds to β-lactoglobulin, clearly showing it to be the predominant component in WPI. The molecular weight of the β-lactoglobulin monomer is 18.4 kDa, however it exists as a dimer in its active state therefore, despite being a slight underestimate, the 33 kDa peak represents the dimer.

The molecular weight of α-lactalbumin (peak 4) was shown to be 15 kDa, which matches theoretical data. An interesting finding is that the height of the RI peak (which measures the concentration) for α-lactalbumin is less than half that of β-lactoglobulin, whereas the height of the UV signal for both of them was very similar. This shows that α-lactalbumin has a higher extinction coefficient than β-lactoglobulin at 280 nm.

The 70k and 150k peaks were assigned to serum albumin and immunoglobulin respectively according to the molecular weight.

All four peaks had narrow PD values less than 1.04, which is as expected due to the fact that protein always has a narrow PD value.

As expected from the separation mechanism, the hydrodynamic radius values decrease with retention volume and confirms that the lower molecular weight proteins have smaller sizes. The intrinsic viscosity values also decrease with molecular weight but not in a linear fashion, revealing the difference in protein structure. This is most clearly illustrated by the intrinsic viscosity of the β-lactoglobulin dimer, which is only 15% higher than the value for the α-lactalbumin monomer, despite being twice the molecular weight. This indicates the structure of the dimeric β-lactoglobulin is more compact than the α-lactalbumin. Aggregation detection

3 Characterization of Whey Protein Isolate (WPI) using multi-detector size-exclusion chromatography APPLICATION NOTE

At a low retention volume of 10-12 mL, the LALS and RALS detectors both showed a single peak, and there were no peaks on the RI or UV detectors at all. The RI and UV signals are proportional to concentration, therefore the concentration of this peak must be very low. Light scattering is proportional to the molecular weight multiplied by the concentration, therefore the molecular weight must be extremely large in order to show a peak in the light scattering plot. Another indication as to the size of the aggregate is the fact that it eluted at a low retention volume. There is also no peak in the viscometer detector. This suggests that these aggregates are very compact and condensed, with a low intrinsic viscosity, as opposed to fibrous or other structure, hence the reason why there is no peak detected by the viscometer detector.

Conclusions The data in this application note show that the Viscotek TDAmax can be used to characterize even complex mixtures of proteins such as those in WPI if they can be well separated by the chromatography. Here, the four components of WPI, immunoglobulin, albumin, β-lactoglobulin and α-lactalbumin were all characterized for their concentration, molecular weight, hydrodynamic size and structure in a single experiment. Some protein aggregates were even identified by the light scattering detector although these were at too low a concentration to fully characterize. The strength of the TDAmax for such applications makes it ideal for food and nutrition applications such as this or for other protein related applications such as the study of biopharmaceuticals.

4 Characterization of Whey Protein Isolate (WPI) using multi-detector size-exclusion chromatography APPLICATION NOTE

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