chromatography (WPI) using multi-detector size … NOTE 3 Characterization of Whey Protein Isolate...
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APPLICATION NOTE Malvern Instruments Worldwide Sales and service centres in over 65 countries www.malvern.com/contact ©2014 Malvern Instruments Limited Characterization of Whey Protein Isolate (WPI) using multi-detector size-exclusion chromatography MOLECULAR WEIGHT MOLECULAR SIZE PROTEIN AGGREGATION Introduction Whey is a by-product of the cheese-making process, and Whey Protein Isolate (WPI) is 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 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
Transcript of chromatography (WPI) using multi-detector size … NOTE 3 Characterization of Whey Protein Isolate...
APPLICATION NOTE
Malvern Instruments WorldwideSales and service centres in over 65 countrieswww.malvern.com/contact ©2014 Malvern Instruments Limited
Characterization of Whey Protein Isolate(WPI) using multi-detector size-exclusionchromatography
MOLECULAR WEIGHT
MOLECULAR SIZE
PROTEIN AGGREGATION
IntroductionWhey is a by-product of the cheese-making process, and Whey Protein Isolate (WPI) isa 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 virtuallylactose free, carbohydrate free, fat free, and cholesterol free.
Whey protein Isolate is of high nutritional value, and has become an importantsource of functional ingredients in various health-promoting foods. It is widely usedin infant formula to provide a natural source of amino acids for optimal growth anddevelopment, 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 rapidlyand 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 presentin 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 threebut the remaining 15% remain allergic. Bovine β-lactoglobulin (BLG) is the majorallergen in cow’s milk, so dairy product manufacturers need to prove the presence orabsence 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, witha molecular weight of 18.4 kDa, and in physiological conditions it exists predominantlyas a dimer.
α-lactalbumin is a protein present in the milk of almost all mammalian species, and isinvolved 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 forsteroids, fatty acids and thyroid hormones in the blood, and it plays a major role instabilizing extracellular fluid volume by contributing to oncotic pressure. Serum albuminis 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 systemto identify and neutralize foreign objects such as bacteria and viruses. In placental
APPLICATION NOTE
2 Characterization of Whey Protein Isolate (WPI) using multi-detector size-exclusion chromatography
mammals there are five antibody isotypes known as IgA, IgD, IgE, IgG and IgM. Atypical Immunoglobulin has a molecular weight of 150 kDa.
In this application note, a sample of WPI was characterized using the ViscotekTDAmax. The 4 main components in this mixture were separated and individuallycharacterized.
Materials and methodsThe samples were analyzed on a Viscotek TDAmax system from Malvern Instruments,with refractive index (RI), ultraviolet (UV), 7°angle light scattering (LALS), 90°anglelight scattering (RALS) and intrinsic viscosity (IV) detectors.
The samples were separated with two Viscotek P-columns (300 x 7.8 mm ID), 1 xP4000 and 1 x P3000, with a protein molecular weight exclusion limit of 700 kDa and1000 kDa respectively.
The mobile phase was 0.1M pH7.0 phosphate buffer, around 500 µg of total materialwas injected for each measurement.The chromatograms were analyzed using the conjugate analysis calculations in theOmniSEC software.
Results and discussionsComponents characterizationThe 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.
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3 Characterization of Whey Protein Isolate (WPI) using multi-detector size-exclusion chromatography
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, correspondsto β-lactoglobulin, clearly showing it to be the predominant component in WPI. Themolecular weight of the β-lactoglobulin monomer is 18.4 kDa, however it exists as adimer in its active state therefore, despite being a slight underestimate, the 33 kDapeak represents the dimer.
The molecular weight of α-lactalbumin (peak 4) was shown to be 15 kDa, whichmatches theoretical data. An interesting finding is that the height of the RI peak (whichmeasures 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 thatα-lactalbumin has a higher extinction coefficient than β-lactoglobulin at 280 nm.
The 70k and 150k peaks were assigned to serum albumin and immunoglobulinrespectively according to the molecular weight.
All four peaks had narrow PD values less than 1.04, which is as expected due to thefact that protein always has a narrow PD value.
As expected from the separation mechanism, the hydrodynamic radius valuesdecrease with retention volume and confirms that the lower molecular weight proteinshave smaller sizes. The intrinsic viscosity values also decrease with molecular weightbut not in a linear fashion, revealing the difference in protein structure. This is mostclearly illustrated by the intrinsic viscosity of the β-lactoglobulin dimer, which is only15% higher than the value for the α-lactalbumin monomer, despite being twice themolecular weight. This indicates the structure of the dimeric β-lactoglobulin is morecompact than the α-lactalbumin.
Aggregation detection
APPLICATION NOTE
4 Characterization of Whey Protein Isolate (WPI) using multi-detector size-exclusion chromatography
At a low retention volume of 10-12 mL, the LALS and RALS detectors both showed asingle peak, and there were no peaks on the RI or UV detectors at all. The RI and UVsignals are proportional to concentration, therefore the concentration of this peak mustbe very low. Light scattering is proportional to the molecular weight multiplied by theconcentration, therefore the molecular weight must be extremely large in order to showa peak in the light scattering plot. Another indication as to the size of the aggregate isthe fact that it eluted at a low retention volume. There is also no peak in the viscometerdetector. This suggests that these aggregates are very compact and condensed, witha low intrinsic viscosity, as opposed to fibrous or other structure, hence the reason whythere is no peak detected by the viscometer detector.
ConclusionsThe data in this application note show that the Viscotek TDAmax can be used tocharacterize even complex mixtures of proteins such as those in WPI if they canbe well separated by the chromatography. Here, the four components of WPI,immunoglobulin, albumin, β-lactoglobulin and α-lactalbumin were all characterizedfor their concentration, molecular weight, hydrodynamic size and structure in a singleexperiment. Some protein aggregates were even identified by the light scatteringdetector although these were at too low a concentration to fully characterize. Thestrength of the TDAmax for such applications makes it ideal for food and nutritionapplications such as this or for other protein related applications such as the study ofbiopharmaceuticals.
APPLICATION NOTE
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