C: Food Chemistry & Toxicology JFS C:Food Chemistry and Toxicology
Determination of Total Protein Content in Gelatin Solutions with the Lowry or Biuret Assay P.Z HOU AND J.M.R EGENSTEIN
ABSTRACT:Gelatins can be obtained from different sources and prepared using different processes,and the end product gelatin may vary in amino acid composition and molecular weight distribution.In the present study,the variation in“protein color”development among gelatins in colorimetric total protein content measurements was investigated at540nm using the Biuret assay and at650nm using the Lowry assay,with bovine serum albumin as the reference protein.In both the Biuret and Lowry assays,the color response varied significantly among gelatins.
The difference in imino acid content was the major factor responsible for this variation,which probably influenced the gelatin helix→coil phase transition and resulted in the difference in gelatin associate state.Based on their “protein color”development abilities in both Biuret and Lowry,gelatins were classified into2major groups with the hierarchical cluster analysis:1group included all cold water fish gelatins,while the other included gelatins from warm water fish,avian,and mammalian species.
Keywords:Biuret,gelatin,hydroxyproline,imino acids,Lowry,protein content
Introduction
G elatinisanimportantfunctionalbiopolymerthatiswidelyused
in foods to improve elasticity,consistency,and stability.It is a
class of proteinaceous substances that are derived from the parent
protein collagen by procedures involving the destruction of cross-
linkages between polypeptide chains of collagen,in some cases,
with some amount of breakage of polypeptide bonds(Johns and
Courts1977).Gelatin can be obtained from mammalian,avian,and
fish species.The principal raw materials used in gelatin production
today are cattle bones,cattle hides,and pork skins.In addition,fish
skin and bone and chicken bone could also serve as the sources of
raw material for gelatin manufacture.Due to the various sources and
manufacture processes of gelatin,the chemical properties of gelatin
may vary in2ways:first,the differences in amino acid composition,
which is similar to that of the parent collagen and,therefore,directly
reflects the influence of species and type of tissue;and second,the
differences in molecular weight distribution,which result from the
variation in the nature or severity of the extraction conditions.
During gelatin or collagen preparation,or its chemical and phys-
iochemical property determinations,the total protein content often
has been monitored by the Lowry assay(Sato and others1986,1987;
Aidos and others1999;Sadowska and others2003),the Biuret as-
say(Young and Lorimer1960;Nomura and others1996;Yoshimura
and others2000;Zhou and Regenstein2004,2005),or the Kjeldahl
method(Montero and others1999;Muyonga and others2004a,
2004b).Although the Kjeldahl method could be applied in the pro-
tein content determination,it is time and sample consuming.It is,
therefore,not the1st choice in a research laboratory for routine de-
terminations(Sapan and others1999).Furthermore,in this method,
although it is well recognized that a factor is needed to calculate the
total protein from the nitrogen content,most data are calculated
using the standard6.25conversion factor counting all nitrogen in
the sample,making it only an approximation of the actual protein
MS20060229Submitted4/24/2006,Accepted8/9/2006.The authors are with
Dept.of Food Science,College of Agriculture and Life Sciences,Cornell Univ.,
Stocking Hall,Ithaca,NY14853Direct inquiries to author Regenstein(E-
mail:jmr9@https://www.doczj.com/doc/094921216.html,).
present.For gelatin,this value was not only very different from other
proteins but also different among different gelatins(Leach and Eas-
toe1977).On the other hand,the Biuret and Lowry assays are2
commonly used colorimetric total protein determination methods,
and both are simple,rapid,and relatively precise(AOAC1990).
With colorimetric total protein determination methods,there are
some factors that may cause a variation in the color response of dif-
ferent proteins and these factors need to be taken into consideration.
In the Lowry assay,factors such as amino acid composition might in-
fluence the development of color(Chou and Goldstein1960;Sapan
and others1999).In the Biuret assay,although the amino acid com-
positionisnotasignificantfactor,theproteinassociationstatemight
result in a variation with respect to the color response(Sapan and
others1999).It has been well known that due to its specific amino
acid composition and protein conformation,the color response of
gelatin was very different from those of other proteins in both the
Lowry and Biuret assays(Gornall and others1949;Lowry and others
1951).The studies on gelatin showed that among gelatins,there were
variations in amino acid composition and molecular weight distri-
bution(Eastoe and Leach1977),which could result in variations in
the“protein color”response among different gelatins.
Thus,the objective of this study is to identify the variation of color
response among gelatins from various sources and preparations us-
ing both the Lowry and Biuret assays,and to determine the main
factors responsible for the variation.
Materials and Methods
Gelatins
Cattle hide gelatin(MCS;SKW Biosystems,Inc.,Waukesha,
Wis.,U.S.A.),pork skin gelatin(MPS;Kind&Knox Gelatine,Inc.,
Sioux City,Iowa,U.S.A.),pork bone gelatin(MPB;Kind&Knox
Gelatine,Inc.),chicken bone gelatin(ACB,Food Industry Technol-
ogy,Miami Beach,Fla.,U.S.A.),and tilapia skin gelatin(FWT;Rous-
selot,Dubuque,Iowa,U.S.A.)were obtained as commercial food
grade gelatins.Catfish skin gelatin(FWC),cod skin gelatin(FCC),
North Atlantic pollock skin gelatin(FCS),and cusk skin gelatin(FCT)
were prepared by an alkaline extraction process.Various Alaska
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doi:10.1111/j.1750-3841.2006.00151.x
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pollock skin gelatins (FCPs)were prepared by different extraction processes to obtain samples with similar hydroxyproline content but different molecular weight distributions:FCP1was prepared by
direct water extraction,FCP2was prepared by an acid extraction process,and FCP3was prepared by an alkaline extraction process
(Zhou and Regenstein 2004,2005).
Colorimetric assays
The color response of gelatins in the Biuret assay was determined at 540nm as described by Gornall and others (1949),while the color
response of gelatins in the Lowry assay was determined at 650nm with a modified Lowry method (Hartree 1972).Bovine serum albu-min (BSA;Sigma-Aldrich,Inc.,St.Louis,Mo.,U.S.A.)was used as
a reference protein in both assays.The concentration of BSA was
determined by using the absorbance at 280nm (Coho and oth-ers 1947;Regenstein and Regenstein 1984),with the absorbance at
320nm serving as background scattering correction.An extinction
coefficient of 0.66(0.1%BSA,1cm)was used.Because there is little
lipid or carbohydrate in gelatins (Gelatin Manufacturers Institute of
America 1993),the protein concentration of gelatins was based on
their dry weight and ash content (that is,dry weight –ash content)
according to AOAC method (AOAC 1990).To identify the variation
of color response among gelatins with the Lowry and Biuret assays,
a reference factor (RF)was applied,which refers to a ratio of the net
absorbance of each gelatin to the net absorbance of BSA (for Biuret,
all net absorbance refers to the determinations at 5mg/mL protein
concentration,while for Lowry,all net absorbance refers to the de-terminations at 0.5mg/mL protein concentration).All experiments
were performed on the original gelatins in triplicate.
Hydroxyproline determination The hydroxyproline content was determined in triplicate by the method of Woessner (1961)with L-hydroxyproline (Sigma-Aldrich,Inc.)as the standard.SDS-polyacrylamide gel electrophoresis (SDS-PAGE)Gelatin solutions (3mg/mL)were diluted 1:2with Laemmli sam-ple buffer (Bio-Rad Laboratories,Hercules,Calif.,U.S.A.)contain-ing 2-mercaptoethanol (50μL in 950μL sample buffer).Samples (the total protein in each sample was 6μg)were then run on SDS-PAGE 7.5%Ready Gels (Bio-Rad Laboratories),together with a broad range of prestained SDS-PAGE standards (myosin,209kDa;beta-galactosidase,124kDa;BSA,80kDa;ovalbumin,49kDa;carbonic anhydrase,35kDa;soybean trypsin inhibitor,29kDa;lysozyme,21kDa;aprotinin,7kDa;Bio-Rad Laboratories).The SDS-PAGE was applied according to Laemmli (1970)using a Mini Protein II unit
(Bio-Rad Laboratories)at a constant voltage of 150V .Protein bands
were stained with Coomassie brilliant blue R-250(Bio-Rad Labo-ratories)and then destained according to the method of Fairbanks and others (1971).
Thermal transformation determinations
The thermal helix-random coil transformation was determined viscometrically by a nonequilibrium method (von Hippel and Wong 1963)with modification.Ten milliliters of gelatin solutions (1mg/mL)in 10-mM phosphate buffer (pH 7)were used for viscos-itymeasurementswithaCanon-Fenskeroutineviscometer(Cannon Instrument Co.,State College,Pa.,U.S.A.).The thermal transition (helix →coil transition)curve (Figure 1)was obtained by measuring the change of viscosity with temperature increasing from 2.5to 50?C;the temperature of the solution was raised stepwise (2.5?C/step)and
maintained for 30min at each temperature before measurements were made.Fractional change of viscosity ={(ηsp )t ?(ηsp )t =50?C )}/{(ηsp )t =2.5?C ?(ηsp )t =50?C )}
The thermal transition temperature (T 1/2)was then determined as the temperature at which the change of viscosity was half com-pleted (Figure 1).Each point is the mean of triplicate measurements.Statistical analysis The regression analysis of the color responses of gelatins in the
Biuret assay and gelatin concentration and the correlation between
the color responses of gelatins and that of BSA in the Lowry as-say were determined with Excel (Microsoft Corp.,Redmond,Wash.,
U.S.A.).The analysis of variance (ANOVA)using the general linear
model procedure and the difference between means using Duncan
test were determined using SAS (SAS Institute Inc.,Cary,N.C.,U.S.A.)
at an αlevel of 0.05.
The correlation between the color responses of gelatins in the
Biuret assay and those in the Lowry assay,the correlation between
the hydroxyproline content of gelatins and their color responses
in the Biuret or Lowry assays,and the correlation between the hy-droxyproline content of gelatins and the thermal transformation
temperature were determined using the rank correlation method
(Rosner 2000),due to the non-normal distribution of the variables.
The Spearman rank-correlation coefficient (r s )was calculated using
Minitab software (Minitab Inc.,State College,Pa.,U.S.A.);the statis-tical significance for r s was further determined by the t -test (Rosner 2000).
The hierarchical cluster analysis was used for searching groups of gelatins among the study ’s variables (the color responses or RF for Biuret and Lowry).Clustering analysis is a classification pro-cedure that involves a measurement of either the similarity or the
distance between objects to be clustered (Massart and others 1988).
Euclidean distance and average linkage were used to compute hier-archical clusters with Minitab software.
curve of gelatin.The thermal transition temperature (T 1/2)was determined as the temperature at which the change of viscosity was half completed.Each point is the mean of triplicate determinations.
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Results and Discussion
Color responses of gelatins in both assays
The statistical comparison of gelatin color responses in protein
assays is better determined at a single protein concentration.How-
ever,to make the results more meaningful,it should be verified
whether the color response ratio between different proteins varies
with changes in protein concentration,especially for the Lowry
method,in which the relationship between color yield and protein
concentration was usually nonlinear(Figure2).To simplify the ex-
planation,only the color response curves of samples FCC and MPS
are shown here,with BSA as the reference protein.
For Biuret,within the range of approximately0.5to5mg/mL
protein concentrations,there was a linear response between pro-
tein concentration and the absorbance at540nm(for all gelatins
and BSA,R2>0.99;Figure2a);further increases in the gelatin con-
centration are not applicable because mammalian gelatins can form
gels at a concentration of around10mg/mL.For the modified Lowry
assay,a linear colorimetric response could not be obtained in the
whole region of0to0.5mg/mL protein concentration(the R2for the
linear colorimetric response of some gelatins was less than0.95).
However,by comparing the color response of gelatins with that of
the Biuret assay and at650nm in the Lowry assay,with
BSA as the reference protein:(a)the Biuret assay;(b)the
Lowry assay.Values are means of triplicate determina-
tions.MPS,pork skin gelatin;FCC,cod skin gelatin;BSA,
bovine serum albumin.
BSA at each given protein concentration,a linear correlation was
observed,which suggested that the color response ratio between
different gelatins and BSA would not vary with changes in protein
concentration(Figure3).Thus,in the Biuret assay and the modified
Lowry method,the comparison of the color responses at1concen-
tration could be used for all protein concentrations under normal
conditions of the assay.The color response for each gelatin was ex-
pressed as a ratio to the net absorbance for BSA at certain protein
content(5mg/mL in Biuret and0.5mg/mL in Lowry),and this ratio
was referred to as the RF(Table1).
The protein-to-protein variation in color response refers to dif-
ferences in the amount of color(absorbance)when various proteins
with the same mass or concentration are determined concurrently
by the same method.The color responses of gelatins showed sig-
nificant variations(P<0.05)in both the Biuret and Lowry assays
(Table1).For each gelatin,the RF for Biuret was higher than that for
Lowry(P<0.05).
Gornallandothers(1949)comparedthe K valuesofanimalgelatin
with other proteins during the Biuret reaction[K=(2–log G)/C
],
gelatins and that of BSA in the Lowry assay.MPS,pork
skin gelatin;FCC,cod skin gelatin;BSA,bovine serum al-
bumin.
Table1---Reference factor(RF)for gelatins in the Biuret
and Lowry assays
RF
Gelatin sources Biuret assay Lowry assay
Cold water?sh gelatins FCP1 1.02±0.01a0.89±0.01a
FCP2 1.04±0.02a0.91±0.01a
FCP3 1.01±0.01a0.89±0.01a
FCC0.99±0.01b0.89±0.02a
FCS 1.02±0.01a0.89±0.01a
FCT0.99±0.01b0.87±0.03a
Warm water?sh gelatins FWC0.90±0.02c0.69±0.01b
FWT0.85±0.01d0.65±0.01c
Avian gelatin ACB0.82±0.01e0.62±0.01d
Mammal gelatins MCS0.83±0.02d e0.62±0.01d
MPS0.83±0.01d e0.63±0.01d
MPB0.84±0.02d e0.64±0.01c d
FCP1,Alaska pollock skin gelatin prepared by direct water extraction;FCP2,
Alaska pollock skin gelatin prepared by an acid extraction process;FCP3,
Alaska pollock skin gelatin prepared by an alkaline extraction process;FCC,cod
skin gelatin;FCS,North Atlantic pollock skin gelatin;FCT,cusk skin gelatin;
FWC,cat?sh skin gelatin;FWT,tilapia skin gelatin;ACB,chicken bone gelatin;
MCS,cattle hide gelatin;MPS,pork skin gelatin;MPB,pork bone gelatin.
All values were mean±standard deviation.The same letter(a,b,c,d,and e)in
the same column indicates no signi?cant differences between means(P<0.05). C476JOURNAL OF FOOD SCIENCE—Vol.71,Nr.8,2006URLs and E-mail addresses are active links at https://www.doczj.com/doc/094921216.html,
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where G is the reading of galvanometer reading for a spectrophoto-metric analysis and C is the protein concentration calculated from a Kjeldahl determination),and suggested that gelatin had a “striking difference ”from most other proteins.The ratio of the K value for ani-mal gelatin to that for human serum albumin (HSA)was about 0.74.However,during their calculation of protein concentration,these authors used a conversion factor of 6.25for animal gelatin,which should have had a value between 5.46and 5.51instead according to Leach and Eastoe (1977).After recalibrating the K value of gelatin using a conversion factor of 5.5,this ratio increased to 0.84,and was similar to the RF values for mammalian gelatins in the present determination (approximately 0.83to 0.84;Table 1),although BSA instead of HSA was used as the reference protein.On the other hand,Lowry and others (1951)also compared the color response of ani-mal gelatin with BSA in their original Lowry determination report.The ratio of the extinction coefficient for animal gelatin to that for BSA was about 0.56.(The extinction coefficient was defined as the optical density at a wavelength of 750nm with 1atom of N per liter;it was 1145for animal gelatin and 2050for BSA.)However,the same problem occurred in the analysis of Gornall and others (1949);that is to say,they applied a conversion factor of 6.25for the animal gelatin.After recalibrating with a conversion factor of 5.5,this ratio increased to 0.63,and was very similar to the RF values for mam-malian gelatins in the present determination (approximately 0.62to 0.64;Table 1).
However,the color responses of fish gelatins,especially gelatins from cold water fish,showed a significant difference from the color responses of mammalian gelatins (Table 1).The color responses of cold water fish gelatin had a value that was almost similar to that of BSA in the Biuret reaction,with an RF ranging from 0.99to 1.04.Similarly,the color responses of cold water fish gelatins were closer to that of BSA compared to mammalian gelatins in the Lowry assay,and the RF value varied from 0.87to 0.91.
The above results suggested that there were variations in color response among gelatins in both the Biuret and Lowry assays.How-ever,it is still not clear what the main reasons for these differences are.Previous studies suggested that gelatins might be different in amino acid composition and/or molecular weight distribution due to their various sources and extraction processes.So the following sections will consider the effect of these 2factors on protein color development.
Effect of hydroxyproline content and molecular weight distribution
The Biuret reaction is almost independent of the amino acid com-position of protein (Sapan and other 1999).However,the association state of protein is important for the color response in the Biuret as-say,because it can influence the opportunities for peptide bonds to react with copper.Gelatin has a super helix structure:each αpolypeptide chain forms a left-handed helix by itself,and 3αchains then form a right-handed super triple helix.When the environmen-tal temperature increases,a helix →coil transition can be observed.The transition temperature of gelatin is influenced by the imino acid content,molecular weight distribution,gelatin concentration,and other environmental conditions.However,among all these fac-tors,the imino acid content (proline and hydroxyproline),espe-cially the hydroxyproline content,was the most significant factor (te Nijenhuis 1997).
Figure 4shows the relationship between gelatin hydroxypro-line content,helix →coil transition temperature and protein color response in the Biuret assay.In addition,the Spearman rank-correlation coefficients (r s )were calculated to determine correla-tions.It suggested that the decreases of hydroxyproline content in
gelatins and their properties.Symbols:open diamond,re-lation between hydroxyproline content of gelatins and their thermal helix-random transition temperatures (T 1/2);closed triangle,relation between hydroxyproline content of gelatins and their color response (RF ,reference factor)in the Biuret assay;closed circle,relation between hy-droxyproline content of gelatins and their color response (RF)in the Lowry assay.
gelatins would decrease the helix →coil transition temperature (r s =0.81,P <0.01),which suggests that the polypeptide chain of gelatin became more flexible and could more easily form complexes with copper ions in the Biuret assay (the r s between the helix →coil tran-sition temperature and protein color response was ?0.79,P <0.01).Thus,the difference in the hydroxyproline content of gelatin is a very important contributor to the variation of protein color response in Biuret (r s was ?0.92,P <0.001).
The color developed in the Lowry assay is believed to arise from the reduction of the phenol reagent by some chromogenic amino acids along with the Biuret reaction (Chou and Goldstein 1960;Wu and others 1978;Sapan and others 1999).The major chromogenic amino acids are tyrosine and tryptophan (Chou and Goldstein 1960).Since gelatin contains no tryptophan and a very little tyrosine (Eas-toe and Leach 1977),the chromogenic amino acids are unlikely to be a major reason for the variations in color response among gelatins.The present study suggests that the hydroxyproline content is a ma-jor factor responsible for the variation of color response in Lowry assay (r s was ?0.94,P <0.001).
Due to the differences in extraction processes,gelatins may also vary in molecular weight distribution.Three Alaska pollock skin gelatin samples (FCP1,FCP2,and FCP3)were prepared using dif-ferent extraction processes.The hydroxyproline content was not significantly different between samples (approximately 7.3%,P >0.05).The gelatin prepared with direct water extraction (FCP1)had the most fractions with a molecular weight lower than 80kDa,while the one prepared with an alkaline extraction process (FCP3)had the most fractions with a molecular weight more than 80kDa (Figure 5).However,for all 3pollock gelatin samples,the RF only varied be-tween 1.01and 1.04with the Biuret and between 0.89and 0.91with the Lowry (Table 1).It suggested that the differences in molecular weight distribution did not affect the color response as much as differences in hydroxyproline content.
Hierarchical cluster analysis Gelatins were clustered on the basis of their color responses (RF ,
Table 1)in the Biuret and Lowry assays.The distance matrix was calculated using the Euclidean distance,and the average linkage
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Vol.71,Nr.8,2006—JOURNAL OF FOOD SCIENCE C 477
C: Food Chemistry & Toxicology was used to evaluate the distance between clusters(Massart and
others1988).
The dendrogram for the classification scheme is shown in
Figure6.Based on their behaviors in the Biuret and Lowry
assays,
Figure5---SDS-PAGE pattern of Alaska pollock skin
gelatins(FCP1,FCP2,and FCP3)prepared with differ-
ent extraction processes.SD represents prestained SDS-
PAGE standards,and the calibrated molecular weights of
these standards were also labeled.FCP1,Alaska pollock
skin gelatin prepared by direct water extraction;FCP2,
Alaska pollock skin gelatin prepared by an acid extrac-
tion process;and FCP3,Alaska pollock skin gelatin pre-
pared by an alkaline extraction process.
tein color”responses(RF,reference factor)in the Bi-
uret and Lowry assays.FCP1,Alaska pollock skin gelatin
prepared by direct water extraction;FCP2,Alaska pol-
lock skin gelatin prepared by an acid extraction pro-
cess;FCP3,Alaska pollock skin gelatin prepared by an
alkaline extraction process;FCC,cod skin gelatin;FCS,
North Atlantic pollock skin gelatin;FCT,cusk skin gelatin;
FWC,cat?sh skin gelatin;FWT,tilapia skin gelatin;ACB,
chicken bone gelatin;MCS,cattle hide gelatin;MPS,pork
skin gelatin;MPB,pork bone gelatin.
gelatins were classified into2major groups:1group included all cold
water fish gelatins studied,while the other included all of the avail-
able gelatins from warm water fish,avian,and mammalian species.
In the1st group,all the cold water gelatins were very similar to one
another.In the2nd group,all but FWC were very similar.Gelatins
from the same species but different tissues(MPB and MPS)were
very similar to each other in both protein assays.
Conclusion
T he Lowry and Biuret assays are colorimetric methods widely
used for total protein determination.The color responses of
gelatins varied significantly in both assays,and the difference in hy-
droxyproline content was the main reason for this variation.Gelatins
were classified into2major groups based on their color development
ability.Thus,for the more accurate determination of total protein
in gelatin solutions with the Lowry or Biuret method,the choice of
the reference standard is important,and only gelatin from the same
source or at least the same hierarchical cluster group should be used
as the standard.This finding most likely can also be extended to the
total protein content determination of collagen,which is the parent
protein of gelatin.
Acknowledgments
This work is sponsored by a grant from NMFS/NOAA of the US Dept.
of Commerce.The authors also thank UniSea,Inc.(Dutch Harbor,
Alaska,U.S.A.)for the support in providing fish skins.
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Vol.71,Nr.8,2006—JOURNAL OF FOOD SCIENCE C 479