PROTEIN DETERMINATION METHODS
1. Kjeldahl Method.
2 Dye binding Method.
3. Biuret Method.
4. Lowry Method.
5. Ultraviolet Method.
1. Kjeldahl Method --- nitrogen determination principles. (1) Digestion
+ conc. H2SO4
+ a catalyst
nitrogen converted into an ammonium ion. (2) Neutralize to get NH3 with NaOH
(3) Steam distillation of NH3 and trap in boric acid.
(4) Titrate with hydrochloric acid. Calculation:
Gram nitrogen/ gram of sample =
*(ml of sample - ml of blank) ¥ N standard acid ´ 0.014g/meq weight of sample
* ml of hydrochloric acid required to titrate sample solution.
1
Disadvantages: not all N is protein.
Purine
Pyrimidine DNA, RNA, etc. Urea
Many plant tissues have > 50% non-protein N.
% N ´ 6.25 = % Protein
6.25 6.38 5.83 5.70 5.30
Corns Milk Whole wheat Wheat flour Nuts
Eggs Barley Peas Oats Meat Rye
Beans Millet
2
2. Dye Binding Method:
Principle: At low pH, basic groups of protein are (+) charged. These will quantitatively bind a (-) charged dye.
What are these basic groups?
+
NH3
CH2
CH2
Lysine
H CH2 N
C NH2
CH2
CH2
H
CH N
N C H
O
O C
C N H
CH2
CH2
CH2
CH
NH2
Arginine
C NH+
HC CH N
H
Histidine
Acid Orange 12:
HO N = N
SO3
Procedure:
1. Mix protein, dye, buffer pH = 2.
2. Filter or centrifuge.
3. Measure optical density (O.D.) of filterate.
3
O.D. dye bound by protein = O.D. dye initial - O.D. filterate
O.D. at 470 nm
Skim milk
6 8 10 12 14 16
% Protein (Kjeldahl)
Factors Influencing Dye Binding determination:
1. Temperature
2. Non-proteins.
3. Buffers systems.
4. Protein quality.
4
3. Biuret Method: Cu++ in alkaline solution form complexity with peptide bonds - give pinkish-purple color.
Measure the intensity of color at 540 nm.
A at 540 nm
% Protein (Kjeldalh)
5
4. Lowry Method: (one of most sensitive methods)
1. Cu++ in alkaline solution to form complexity with protein.
2. Cu++ catalyses oxidation of phenol group of tyrosine with phosphomolybdic-phosphotungstic acid.
A at 750 nm
m g 0f protein (Kjeldalh)
5. Ultra-violet Absorption (UV) at 280 nm
1. Chromophoric side chains of aromatic amino acids (Trosine, Tryptophan).
2. Absorption at 280 nm. “Non-destructive means to determine protein”.
3. Calculation protein conc. based upon absorption.
6
6. Fluorescence Method:
Tyrosine is a fluorescent compound. Tryptophane is a fluorescent compound. Excite the amino acids at 280 nm. Measure emission at 348 nm.
Advantage: more sensitive than UV absorption.
Intensity of Emitted Fluorescence
at 348 nm
mg of protein/ml of solution
What is fluorescence and how to measure it?
Excited State Emits radiation (fluorescence) Decay yields fluorescence at
longer wavelength
Ground State
By using specific l (wavelength) to excite and measure output at a specific l. It is rather specific.
Problems:
Turbidity/Quenching (self or others)/Expensive/Quantitation is difficult.
7
Amino Acid Determination:
A. Hydrolysis
1. Overnight in 6 M HCl at 1000C.
2. Enzymes.
B. Separation by ion exchange.
8
MECHANISM OF ION-EXCHANGE CHROMATOGRAPHY OF AMINO ACIDS
SO3
Na+
-
H3N Na+
+
COOH OH
+
pH 2
So3
Exchange Resin
H3N
COOH
SO3
H3N+
-
COOH
OH
pH3.5
So3
H3N+
Na+
Na+
COO-
H+ OH- = H O
SO3
H3N
COO-
H+ OH- = H2O
3 Na+
pH4.5
9
Moles/Liter
VAL
ALA
LYS
HIS
ASP
GLU
LEU
pH 2.25 pH 3.25 pH4.25
10
Some Important Reactions of Proteins
Denaturation
Changes in 2o, 3o, 4o structure. By heat.
Heavy metals (Hg is most common).
pH (trichloroacetic acid, phosphotungstic acid) Salt (NaCl or ammonium sulfate [NH4]2 SO4)
Reasons for Precipitating Proteins
1. Purify, concentrate protein.
2. Remove protein which cause: turbidity/emulsion/troublesome.
Manifestation of Denaturation
1. Decreased solubility.
2. Alteration of size and shape
3. greater reactivity
4. Decreased biological activity (enzyme + immune proteins)
5. Increased sensitivity to electrolytes.
6. Nutritive value.
What is essential amino acids?
Amino acids which the body cannot make (or make enough of) for protein synthesis due to lack of enzymes.
11
Essential Amino Acids: Histidine Isoleucine Leucine
Lysine Methionine Phenylalanine Threonine Valine
Limiting amino acid is the essential amino acid which is lacking in the protein to have a balanced protein.
Product Limiting Amino Acid
Corn Lysine Oats Lysine Rice Lysine Wheat Lysine Sesame Seed Lysine
Cow’s Milk Methionine Potato Methionine Chick Pea Methionine Green Pea Methionine Cotton Seed Isoleucine Beef Valine
Chicken Tryptophan
12
PROTEIN QUALITY DETERMINATION
1. Protein Efficiency Ratio.
2. Biological Value.
3. Net Protein Utilization.
What are the measurements of protein quality?
For labeling purposes, one needs to know the protein efficiency ratio.
1. If PER e casein (2.5), the RDA = 45 g/day.
2. If 0.5 < PER < 2.5, then RDA = 65 g/day.
3. If PER < 0.5 (20% of casein), then “not a significant source of protein”.
How does one determine PER?
1. Male lab rats e 21 days, £ 28 days of age, at least 10 rats/group.
2. Feed a standardized diet containing salt mix, vitamins, cotton seed oil, cellulose, starch or sucrose + water for 28 days.
3. Measure weight gain and food intake at regular intervals, not > 7 days.
4. PER = Weight Gain/Gram of Protein in Diet.
5. Usually normalized for casein = 2.5.
6. Determine protein quality of sample as ratio of sample PER to reference casein
PER.
Protein Efficiency Ratio = Gain in weight per gram protein eaten.
13
Protein efficiency ratio is a number that descries how well a given protein supplies the building blocks for rapid growth.
Product PER
Rice 100% 2.30
Rice 70% Black Beans 30% 2.70
50% 50% 2.60
20% 80% 1.30
100% NIL
Corn
+ 0.4% Lysine
+ 0.07% Tryptophan 2.14
Corn (50%) + Black Beans (50%) 2.05
Percentages refer to the percent of the protein supplied by that source.
14
Product PER
Soybean 2.32
Cotton Seed Meal 2.25
Egg 3.90
Sesame Seed 1.77
Chick Peas 1.68
Peanuts (ground nuts) 1.65
Kidney Beans 0.88
OTHER PROTEIN QUALITY DETERMINATION Biological Value (BV)
Net Protein Utilization (NPU)
BV = Retained Nitrogen (nitrogen intake - fecal & urinary nitrogen)/Absorbed Nitrogen
(nitrogen intake - fecal nitrogen)
NPU = Retained Nitrogen/Intake Nitrogen = BV ´ Digestibility
Analytica Chimica Acta 433 (2001) 155–163
Determination of protein content using a solid phase spectrophotometric procedure
L.F. Capitán-Vallvey a,∗, O. Duque b, G. Mirón-Garc´ıa a , R. Checa-Moreno c
a Department of Analytical Chemistry, University of Granada, 18071 Granada, Spain
b IMRE, Universidad de La Habana, Havana, Cuba
c Laboratorio Nacional de Sanidad y Producción Animal, Ministry of Agriculture, Fisheries and Food, 18320 Santa Fe, Granada, Spain
Received 16 June 2000 ; received in revised form 18 October 2000 ; accepted 25 October 2000
Abstract
We used a conditioned fiberglass paper to perform the reaction and to retain the reaction products of the Lowry method for protein content. The analytical parameter was obtained by measuring directly the absorbance of the support. Different influencing chemical, support related and instrumental variables were studied and optimized. The usual asymptotic behavior of the analytical function response was improved here by linearizing using a quadratic function. Using 10 ml of sample, the applicable concentration range was between 25 and 200 mg l−1 , with a detection limit of 17 mg l−1 and a R.S.D. around
5%. The method was applied to the determination of protein in eggwhite samples coming from different birds. The method proposed here for protein content shows higher sensitivity than solution methods, less dependence on protein nature and higher stability of the species fixed on the support. © 2001 Elsevier Science B.V. All rights reserved.
Keywords: Protein determination; Lowry procedure; Glass paper; Solid phase procedure
1. Introduction
The method of Lowry et al. [1] is probably one of the most widely used procedures for the quanti- tative determination of protein in a simple, sensitive and precise way. Nevertheless, this procedure has sev- eral drawbacks coming from the lack of specificity, the high number of interferences, slow reaction rate, instability of some reagents and nonlinearity of the calibration function [2].
The use of solid supports (cellulose, ion-exchange resin loaded paper or PTFE, chelating paper, fibe glass, etc.) is a very elegant way to retain analytes, and it
∗ Corresponding author. Tel.: +34-958-243326;
fax: +34-958-248436.
E-mail address: lcapitan@ugr.es (L.F. Capita´n-Vallvey).
is often used as a preconcentration procedure. When the sample is concentrated by fixing it on the sup- port, it is usually desorbed with the aid of one reagent or dissolved together with the filter, before making the analytical measurements. It is also possible to make the measurement directly on the filter in order to combine together retention and measurement [3]. The UV–VIS absorption techniques used in connec- tion with the above-mentioned solid phase retention systems are related to solid phase spectrophotometry (SPS) [4].
In this paper, we study the Lowry method for pro- tein content using the methodology of SPS with the aim of simplifying the procedure and improving its characteristics. The goal of this work is to explore the use of SPS in the analysis of protein content, with- out preconcentrating the reaction product, and only as
0003-2670/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S0003-2670(00)01269-1
156 L.F. Capita´ n-Vallvey et al. / Analytica Chimica Acta 433 (2001) 155–163
a medium to practice the Lowry reaction and later to measure the optical property directly on this support. The determination of proteins based on the reduc- ing character of some aminoacid constituents in the face of phosphomolibdic or phosphomolybdo-tungstic reagent in a solution has been studied at great length and used in many different forms; however, the de- termination over a solid support could contribute advantages regarding the stability of the result of the reaction, sensitivity, and a lesser dependence on the
protein nature.
2. Experimental
2.1. Apparatus and software
All spectrophotometric measurements were per- formed with a single beam UV–VIS Beckman DU-8B spectrophotometer. A pellet holder (ref. MD-OZ) from an infrared spectrophotometer Beckman IR 4240 was used to allow the UV–VIS absorbance measure- ment for the different supports studied. A microplate reader Multiskan MS 3.0 Labsystems (Helsinki, Fin-
land) was used for the Bio-Rad® DC protein assay
was used as a reference method.
A Crison digital pH-meter with a combined glass-saturated calomel electrode, and a micropipette Biohit Proline microtip 1–10 and 10–50 ml in tray were used. Also a dry freezer Dura Dry from FTS Systems (Stone Ridge, NY, USA), a microwave oven
850 W ref. Complete 220 from UFESA (Barcelona, Spain) and a infrared heat lamp (250 W) were used.
Software programs used for the statistical treatment of the data were Statgraphics Plus for Windows 3.1 software package (Statistical Graphics Corporation, USA, 1994–1997) and Excel software package from Microsoft Office 97, version 8.0, 1997.
2.2. Reagent
All reagents used were of analytical reagent grade unless stated otherwise. Reverse-osmosis type quality water (Milli-RO 5 Plus and Milli-Q 185 Plus station from Millipore) was used for preparation of reagents. Protein stock solution (1000 mg l−1 ) was prepared
by dissolution of 1.0000 g of BSA (from Sigma- Aldrich Quimica, Madrid, Spain) in 1 l of 0.1 M
H2 PO4 −/HPO4 2− pH 9 buffer solution. Working so- lutions were prepared by dilution in the same buffer solution and were stored at 4◦C for at least 2
months. Bio-Rad® DC Protein Assay reagents (A
(alkaline copper tartrate solution) and B (Folin reagent)) (Bio-Rad, CA, USA) were used. Sodium dodecylsulphate, benzethonium chloride, sodium
1-heptanesulphonate, methyltrioctylammonium chlo- ride and hexadecyltrimethylammonium bromide, all from Sigma, were also used.
Different types of filter paper were tried as solid supports. These papers were cut into a circular shape of 16 mm diameter. Conventional cellulose paper from Albet (ref. 235) (Albet, Barcelona, Spain) and Whatman No. 4 (Whatman, Maidstone, UK), cellu- lose phosphate cation exchanger paper (Whathman, P-81), anion exchanger paper (Whatman, D-81) func- tionalized with amino groups, polyethylene coated paper (Albet, ref. 255 P.E.), glass-fibre filters (Albet, ref. FV-A, FV-B, FV-C, FV-D, FV-F and FV-G), and quartz-fibre filters (Albet, ref. FQ-A) were used.
2.3. Absorbance measurements
In order to avoid a lack of homogeneity in the paper, each measurement was performed against the same support without added reagents. Next, the pa- per support with the reaction products retained was placed on a 13 mm path light KBr pellet holder from an infrared spectrophotometer and subsequently at- tached into the sample compartment of a UV–VIS spectrophotometer. The absorbance was measured at
750 nm with a slit width of 5 nm and a read average of 10. The net absorbance of the product fixed on the paper disk was obtained at 750 nm subtracting the background absorbance of a paper disk equilibrated with blank solution.
2.4. Procedure
Benzethonium chloride (BZ) solution (10 ml, 0.3%) was added to a Albet FVG fiberglass support and the support was dried for 45 s. at medium potency (around
400 W) in a microwave oven. To proceed with the anal- ysis, 10 ml of sample containing 0.2–3.0 mg of protein were added, then 10 ml of phosphomolybdo-tungstic reagent were added and there was a wait of 1 min. Next, 10 ml of 1:20 Folin reagent diluted with water
L.F. Capita´ n-Vallvey et al. / Analytica Chimica Acta 433 (2001) 155–163 157
were added and 5 min passed before drying again at around 400 W in a microwave oven. Another paper
disk was prepared by using 0.1 M H2 PO4 −/HPO4 2−
pH 9 buffer solution as a blank and treated in the same
way. Both absorbances, from sample and blank filter papers, were measured as described in absorbance measurements. The calibration curve was drawn in the same way using BSA solutions of known concen- tration.
2.5. Preparation and treatment of the samples for analysis
The samples analyzed here consisted of eggwhites coming from different bird species, namely, greylay goose (Anser anser), turkey (Meleagris gallopavo), peacock (Pavo cristatus) and pigeon (Columba livia) [5]. A homogenized pool of egg white from several eggs of each of the above species was aliquoted into
2 ml vials and lyophilized at −80◦C over 24 h. Ade-
quate amounts of lyophilized egg white samples were dissolved in 0.1 M H2 PO4 −/HPO4 2− pH 9 buffer solu- tion and they were analyzed for protein content by the
proposed method and by the solution Lowry method supplied by Bio-Rad® and used as a reference.
3. Results and discussion
We selected different solid supports to perform this study. Those were cellulose conventional filter paper, cellulose cation exchanger paper containing phosphate as functional groups, cellulose anion exchanger paper with amino as functional groups, polyethylene coated paper, glass-fiber paper and quartz-fiber paper. It was not possible to use the cellulose-type support because of its reaction with the Folin reagent and its giving off of high signals of the blank, although with the Whatman 4 paper the signal is 40% higher than the Albet paper. The polyethylene paper shows a similar behavior, since it also contains a layer of cellulosic support. A reaction is not produced over the cation exchanger paper, while a signal is given off by the anion exchanger paper, which is 200% higher than with the Albet cellulose paper. Nevertheless, with all of the cellulose basis papers, the blanks show a high signal on the order of that which the protein generates in the working range concentrations, such that it makes
their use impossible, because of the low sensitivity and inability to reproduce what they would give off.
On the contrary, the fiberglass filters, which do not contain any type of binder and whose primary useful- ness is the retention of solid particles in liquids and gases, produce a good result. They allow for the re- action, a more homogeneous dispersion of the results of the reaction than with the previous solid supports, and some very low values of the blanks, due to the absence of reducing properties. The quartz-fiber filter behaves in an analogous way, with a smaller output in the reaction and less stability, although the blanks are better. Consequently, the glass paper support was cho- sen. of the different types available, the Albet FVG
(0.28 mm, weight 65 g m−2 ) was chosen, as it gives
off a smaller background signal, being less thick.
3.1. Influencing variables
To establish the optimum conditions for the pro- duction of a useful analytical signal, a series of ex- periments were conducted, and compromise values of the experimental variables were selected. Vari- ables influencing the system can be divided into three groups: chemical, support related and instrumental variables.
3.1.1. Chemical and support related variables
The results of the reaction that originates with the protein does not spread uniformly over the selected support, but concentrates in the form of a halo on the edges of the fiberglass support, since the solution in which it originates does not wet the support in a homogenous manner. This means a considerable lack of reproducibility in the measurements. It was proved that if a surfactant is present in the medium of reac- tion, it evened the distribution of the product over the support and, consequently, the reproducibility of the measurements.
As surfactants we tried sodium dodecylsulphate (SDS), BZ, sodium 1-heptanesulphonate (HS), methyltrioctylammonium chloride (MTOA) and hex- adecyltrimethylammonium bromide (HDTMA). BZ and HDTMA gave the best results (265% BZ, 261% HDTMA, 126% HS, 123% SDS and 120% MTOA above the signal without the presence of a sur- factant). BZ was chosen as the surfactant to work with because, although HDTMA gives off signals of
158 L.F. Capita´ n-Vallvey et al. / Analytica Chimica Acta 433 (2001) 155–163
Fig. 1. Influence of drying time on solid support absorbance. Dash line: microwave oven heating. Discontinuous line: infrared lamp heating. Three replicates per point.
similar intensity, they are less reproducible, and give rise to a greater swelling of the fiberglass membrane, a phenomenon observed in all of the surfactants tested. The way in which the surfactant made contact with the medium of the reaction was studied, with three pos- sibilities tried: (i) as an additional reagent, (ii) added to the solution that contains the protein problem; or (iii) through an earlier immersion of the support in the (BZ) solution. The third possibility does not deliver good results because it brings about a greater swelling of the support, even through the signal is high; with the addition of BZ to the protein solution, the reaction over the support is not produced. For this reason, the
first option was chosen.
It was observed that the protein solution spreads in a radial manner over the support if it has been wet with the surfactant solution, with a consequent reduc- tion of the signal. To prevent this, we propose drying the support after the addition of the surfactant and be- fore the addition of the protein. Drying by the forced passage of air through the filter (for a concentration of
200 mg l−1 of protein the absorbance is 0.237±0.031),
by heating below an IR lamp (0.122 ± 0.023), or by heating in a microwave oven (0.327±0.040) were tried (Fig. 1). Drying in a microwave oven was selected, as it produces the best signal with the least treatment time, although with less reproducibility.
In Fig. 2, the influence of the concentration of BZ is observed. The maximum signal was observed between
0.2 and 0.8%, decreasing on both sides of this interval. For the working concentration, 0.3% was chosen. The volume of BZ used for the treatment of the support was influential. If the volume added is less than 5 ml, the area of the stain created is too small to produce a reading that can be reproduced; while if it is larger than 20 ml, the subsequent drying after the addition of the rest of the reagents creates a swelling of the support and makes the signal even more difficult to reproduce. For this reason a volume of 10 ml of 0.3% BZ was used.
Having demonstrated the necessity of using BZ to condition the support, we turned to the study of the variables that influence the development of color on this support using the Lowry method for the determi- nation of proteins.
As it is not our objective to study again variables sufficiently known and described [2] that affect the Lowry method (such as the concentration of the cupri-tartaric reagent or the waiting time before the addition of the Folin reagent), this study was carried out in conjunction with those variables specific to the solid phase procedure (such as how to dry the support after the addition of BZ or the heating of the support for the development of color), with the objective of choosing those statistically significant variables and working towards their optimization.
Before carrying out the screening of variables, it was necessary to define the variable space in which
L.F. Capita´ n-Vallvey et al. / Analytica Chimica Acta 433 (2001) 155–163 159
Fig. 2. Influence of benzethonium chloride concentration on absorbance.
the response had to be investigated; this selection was based on previous experiments. The order of the ex- periments was randomized.
Variables selected for study were: BZ concentra- tion (A) (at 0.1 and 0.4% levels), support drying time (B) (at 1 and 2 min levels), dilution of cupri- tartaric reagent (Bio-Rad A) (C) (at 1:10 and 1:40 lev- els), delay time before Folin reagent addition (D) (at
5 and 10 min levels), lifetime of the Folin reagent (E) (at 1 and 45 min levels), dilution of phosphomolybdo- tungstic reagent (Bio-Rad B) (F) (at 1:10 and 1:500 levels), color development time (G) (at 5 and 15 lev- els) and final drying time (H) (at 1 and 3 min level).
We had eight factors to study and two response vari- ables: the protein and the blank measured absorbances. The influence of these factors was investigated by a two-level factorial design, so we performed a factor
screening running a sixteenth fractional design (28−4 )
to select influential factors in a first step. The statis-
tically significant factors were optimized in a second step [6].
Fig. 3 indicates that there are two statistically sig- nificant factors: support drying time and delay time before Folin reagent addition. Next we performed the optimization of the above two variables through a response surface design. We observed that a signal maximization of both factors led to a blank signal in- crease (Fig. 4). So, we accepted as appropriate values
45 and 90 s. for support drying time (B) and for delay time to adding the Folin reagent (D), respectively.
Fig. 3. Standardized Pareto chart for screening variables.
Fig. 4. Estimated response surface. (A) Protein; (B) blank.
160 L.F. Capita´ n-Vallvey et al. / Analytica Chimica Acta 433 (2001) 155–163
3.1.2. Instrumental variables
When measuring on a nonhomogeneous solid sup- port, the measurement conditions to obtain repro- ducible results are very important; in this case, due to the high absorption and dispersion characteristics of fiberglass paper used as a support. To this end, the ideal instrumental conditions to carry out the reading were studied. As variables for study we selected read average (between 10 and 99) and slit width (between
0.2 and 5.0 nm) and as responses: absorbance, mea- surement precision and data acquisition time. The de- sign performed to optimize those parameters showed a maximum signal at a higher slit width, whereas the read average was not significant. The use of a read average of 10 and a slit width of 5.0 nm gave the best response, minimizing the acquisition data time and improving the measurement precision.
3.2. Calibration and analytical features
The calibration function for the determination of protein following the Lowry method shows a curving line that is difficult to adjust to a linear model. It has been proved that this deviation of linearity is inher- ent in the reaction mechanism [2]. The determination of protein over a solid support studied here exhibits an asymptotic behavior similar to the Lowry solution method.
The calibration function for Lowry’s method has been widely studied in literature, finding a great variety of applied models and techniques, from a
single least squares regression to a more complex and laborious iterative process, such as the exponential and logarithmic models proposed [7]. In this work, we checked the fitness of several models, some of them proposed in the literature.
Table 1 shows the parameter obtained for each model tested. Parameters a, b and c from models (9), (10) and (11) cannot be obtained through analytical equations coming from least squares development. Therefore, we used an iterative model [7], which de- pends on the properties of the model. If the value of c is fixed, we can obtain expressions for the parameters a, b and sum of squares for error (SSE), as a function of c. Therefore, the iterative procedure consists of using several values for c and minimizing SSE:
n
SSE = X(Yi − Yˆi )2
i=1
Similarly the rest of the models proposed have been studied using the same criterion. Between the differ- ent models studied, models (2), (4) and (8) shows the best results. Model (11) could be considered as an extension from model (8), adjusted to a higher degree (1/0.15 = 6.66). As these three models offer similar results, we prefer to use model (8) as the calibra- tion function for the proposed method because it is less complex, offering similar results. A calibration graph for the determination of protein was prepared as described under procedure using BSA as standard. The best regression line equations for the protein
Table 1
Calibration models tested
Model Parameters R2 (%) SSE LOF (%)
a b c
y = a + bx (1) 0.009 0.00200 – 93.85 0.0270 0
y = a + b ln x (2) −0.465 0.14600 – 98.98 0.0111 26
y = a + b√x (3) −0.135 0.03300 – 98.14 0.0150 1
y = a + b√x + c(√x)2 (4) −0.241 0.05800 −0.001 99.15 0.0105 40
y = a + bx + cx2 (5) −0.058 0.00345 −0.000008 98.69 0.0130 5 ln y = a + b ln x (6) −5.410 0.81500 – 97.84 0.0710 3 y = (a + b ln x)2 (7) −0.406 0.42500 – 98.46 0.0160 9
y = a + b√4 x (8) −0.424 0.19830 – 99.08 0.0090 33
y = a + b e−cx (9) 0.380 −0.45780 0.01 99.06 0.0021 8 y = a + b ln(x + c) (10) −0.590 0.16900 10 99.06 0.0017 18 y = a + bxc (11) −0.820 0.51100 0.15 99.06 0.0017 21
L.F. Capita´ n-Vallvey et al. / Analytica Chimica Acta 433 (2001) 155–163 161
Table 2
Analytical parameters of proposed method model y = a + b√4 x
Parameter Value
Intercept (a) −0.4247
Slope (b) 0.1983
Table 3
Determination of protein in different types of lyophilized eggwhite using the Lowry solution as a reference methoda
Sample Bird Determinated protein (%)
Correlation coefficient (r) 0.996
Lack-of-fit test (P-value) (%) 33.0
Reference
method
Proposed
method
Dynamic range (mg l−1 ) 25–200
Detection limit (mg l−1 ) 17
Quantification limit (mg l−1 ) 20
Precision (R.S.D. (c) %) 13 (25 mg l−1 )–5 (200 mg l−1 ) Sensibility (SSE/b) (mg l−1 )a 20
SSE 0.009
a The minimum concentration increase possible; SSE: sum of squares for error.
concentration is indicated in Table 2 in the range
25–200 mg l−1 , whereas we can extend the range un- til 800 mg l−1 (Fig. 5) but the precision decreases, as indicated in its heteroscedastic behavior.
The proposed method is approximately twice as sen- sitive as the solution method, as we can see in Fig. 6. On the other hand, the behavior of the three proteins assayed as standard, albumin, casein and collagen, is very similar. This is a significant difference with re- spect to the solution method.
The analytical parameters are summarized in
Table 2. The relative standard deviations were 13 and
5% for nine replicates for the determinations at 25 and 200 mg l−1 protein level, respectively.
Greylay goose Anser anser 90 104
Turkey Meleagris gallopavo 95 84
Turkey Meleagris gallopavo 96 83
Peacock Pavo cristatus 102 111
Dove or pigeon Columba livia 89 68
a Each datum is the average of n = 3–6 determinations.
3.3. Validation and application
The proposed method was applied to the determi- nation of protein in five different lyophilized eggwhite samples coming from different birds. As a reference method, we used Lowry’s solution method supplied
by Bio-Rad®. The calibration model used for the ref-
erence method was studied, finding that both linear and quadratic functions best fit the experimental cali- bration data in the same range as the proposed method (25 to 200 mg l−1 ). For purposes of comparison, we used the quadratic model here.
Table 3 shows the results obtained when the pro- posed method was applied to five samples with six replicates. The results concluded that both methods
Fig. 5. Calibration graph for protein.
162 L.F. Capita´ n-Vallvey et al. / Analytica Chimica Acta 433 (2001) 155–163
Fig. 6. Calibration graphs for different protein standards. (A) Albumin; (B) casein; (C) collagen. Wide point: solution method; filled point:
proposed method.
L.F. Capita´ n-Vallvey et al. / Analytica Chimica Acta 433 (2001) 155–163 163
are statistically comparable when using the proposed method and the reference method, using a paired t-test. In both cases we accepted the null hypothesis (they are not significantly difference) for the t-test at a 95% probability level (P = 30).
4. Conclusions
A new methodology for protein determination based on Lowry’s method is proposed based on the use of fiberglass as a medium to carry out the reaction and measurement. The colored species coming from the reaction are fixed over the preconditioned support. The posterior spectrophotometric measurement of the solid phase allows for the protein determination. The proposed method has these advantages: higher sensi- tivity than with the solution method, less dependence on protein nature, higher stability of the species fixed on the support (more than 2 years without apparent loss in measurement value (<5%)), and as a disad- vantage a lesser reproducibility than with the solution method.
Acknowledgements
This study was funded by the General Subdirec- torate for Training and Knowledge-sharing of the Spanish Ministry of Education and Culture (Project No. PB98-1302). We thank the Laboratorio Nacional de Sanidad y Producción Animal. Ministry of Agricul- ture, Fisheries and Food, (18320) Santa Fe. Granada (Spain) for their support.
References
[1] O.H. Lowry, N.J. Rosenbrough, A.L. Farr, R.J. Randall, J.
Biol. Chem. 193 (1951) 265.
[2] G.L. Peterson, Anal. Biochem. 100 (1979) 201.
[3] F. Capitán, R. Checa, R. Avidad, L.F. Capitán-Vallvey, Talanta
42 (1995) 711.
[4] L.F. Capitán-Vallvey, R. Avidad, M.D. Fernandez-Ramos, Recent Dev. Pure Appl. Anal. Chem. 1 (1998) 149.
[5] R. Peterson, R. Mountfort, G.P.A.D. Hollom, Guia de campo de las aves de España y de Europa, Omega, Barcelona, Spain,
1980.
[6] G.E.P. Box, W. Hunter, J.S. Hunter, Estad´ıstica para investigadores, Editorial Reverté, Barcelona, Spain, 1993.
[7] W. Bates, D. McAllister, Anal. Biochem. 59 (1974) 190.
United States Department of Agriculture
Food Safety and Inspection Service, Office of Public Health Science
CLG-PRO4.03 Page 1 of 8
Title: Protein Determination by Combustion
Revision: 03 Replaces: CLG-PRO4.02 Effective: 07/27/2009
Contents
A. INTRODUCTION ..................................................................................... 2
B. EQUIPMENT ........................................................................................... 2
C. REAGENTS AND SOLUTIONS ............................................................... 3
D. STANDARDS........................................................................................... 3
E. SAMPLE PREPARATION ........................................................................ 4
F. ANALYTICAL PROCEDURE ................................................................... 4
G. CALCULATIONS ..................................................................................... 4
H. SAFETY INFORMATION AND PRECAUTIONS ...................................... 5
I. QUALITY ASSURANCE PLAN ................................................................ 6
J. WORKSHEET.......................................................................................... 7
K. APPROVALS AND AUTHORITIES .......................................................... 8
United States Department of Agriculture
Food Safety and Inspection Service, Office of Public Health Science
CLG-PRO4.03 Page 2 of 8
Title: Protein Determination by Combustion
Revision: 03 Replaces: CLG-PRO4.02 Effective: 07/27/2009
A. INTRODUCTION
1. Theory
Total protein is determined using nitrogen analysis. The sample is combusted with oxygen and the gases containing nitrogen oxides are collected in a ballast tank until a specified pressure is reached. Helium is used as a carrier and an aliquot of combustion gas containing nitrogen oxides is reduced to nitrogen. It is then passed through a tube containing magnesium perchlorate and sodium hydroxide on a silicate carrier to remove water and carbon dioxide. The nitrogen is measured with a thermal conductivity detector using helium as a reference. Nitrogen is then converted to protein using a conversion factor.
Note: This method is not an endorsement by the Food Safety and Inspection Service
(FSIS) of the LECO FP-2000® over other commercially available instruments.
It may be necessary to use operating procedures and/or follow manufacturer's instructions for equivalent instruments from other manufacturers.
2. Applicability
This procedure is applicable to the determination of protein content in fresh and processed meat and poultry products.
B. EQUIPMENT
Note: Equivalent instrumentation or apparatus may be substituted.
1. Apparatus
a. Robot Coupé food processor - Robot Coupé U.S.A. Inc. b. Analytical balance - capable of weighing to 0.1 mg.
c. Forced draft oven - Adjustable to 101 ± 1 °C.
d. Three two-stage compressed gas regulators - Each set at 40 psi. e. Ceramic combustion boats - Cat. No. 529-203, LECO.
f. Foil Boat liners for liquid samples - Cat. No. 502-343, LECO.
2. Instrumentation
a. LECO FP-2000 Protein Analyzer - Version 4.08 or equivalent software, and Autoloader. (Instrument parameters must be optimized for specific instrumentation used). The following are examples of settings for the LECO FP-
2000:
Furnace temperature: 1150 °C.
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CLG-PRO4.03 Page 3 of 8
Title: Protein Determination by Combustion
Revision: 03 Replaces: CLG-PRO4.02 Effective: 07/27/2009
Lance flow: 1.0 L/min. Purge flow: 4.5 L/min. TC cell sensitivity: 1500.
Nitrogen conversion factor: 6.25 for meat and meat products. b. Printer: 9-pin, Okidata Microline 320.
C. REAGENTS AND SOLUTIONS
Note: An equivalent reagent or supply may be substituted.
3. Reagents and supplies
a. N-Catalyst - Cat. No. 502-049, LECO.
b. Anhydrone (Magnesium Perchlorate) - Cat. No. 501-171. LECO.
c. Lecosorb (Sodium Hydroxide on silicate carrier) - Cat. No. 502-174, LECO. d. Silicone grease - Cat. No. 501-241, LECO.
e. Leak detection solution - Cat. No. 502-213, LECO. f. Copper Sticks - Cat. No. 502-304-500, LECO.
g. Copper Turnings - Cat. No. 501-621, LECO.
h. Glass wool for furnace filter packing - Cat. No. 501-081, LECO. i. Steel wool - Cat. No. 502-310, LECO.
j. Cylinder - Compressed air, medical quality.
k. Cylinder - Oxygen, 99.99% purity, Airco 4.4 grade. l. Cylinder - Helium, 99.99% purity, Airco 5.0 grade.
D. STANDARDS
1. Combustion Calibration EDTA
Approximately 99.5% Pure, Cat. No. 25, 404-5, Aldrich Chemical Company, or other suitable organic material of high purity and known nitrogen content.
Determine the % meat protein equivalent of the standard by multiplying the % purity by
59.91 (6.25 x %N in EDTA (9.586%).
Note: A standard curve must be established for each method (see LECO manual). The drift of the curve can be corrected as often as needed by analyzing three or more EDTA standards, and using the drift correction icon.
United States Department of Agriculture
Food Safety and Inspection Service, Office of Public Health Science
CLG-PRO4.03 Page 4 of 8
Title: Protein Determination by Combustion
Revision: 03 Replaces: CLG-PRO4.02 Effective: 07/27/2009
E. SAMPLE PREPARATION
Process the sample until a homogeneous mixture is obtained.
F. ANALYTICAL PROCEDURE
1. For the operation of LECO FP-2000
Prepare instrument by following the procedure outlined in the operator’s instruction manual (i.e. pack reagent tubes, perform leak checks, etc.).
a. Weigh 1.0 ± 0.2 g of sample into a ceramic boat.
b. Dry samples in a 101 ± 1 °C convection oven for 45 ± 5 min. After drying, place in desiccator to cool and/or hold until ready to load into the instrument.
Note: Sample may be stored in desiccator until analyzed.
2. LECO FP-2000 setup
a. Run 5 or more blanks until values are reproducible and lower than 0.375%
protein. Drift correct for the blank using the last three consecutive values.
b. Run 4 or more EDTA standards until three consecutive values agree within
< 0.15% protein of each other. Use the last three consecutive values to drift correct for the EDTA .
3. Sample Analysis
a. Load the set of samples into autoloader. (For quality control, an EDTA standard should be placed for every eight samples. If the instrument drifts during the run, these EDTA standards can be used to drift correct and samples can be recalculated.)
b. Enter standard and meat sample weights in the order in which they are in the rack. Weights can be entered manually, from an interfaced balance, or electronically (from floppy disc).
c. Analyze samples.
G. CALCULATIONS
Calculations are done automatically by the data system. The results will be reported as
% nitrogen unless a nitrogen factor of 6.25 (for meat) was entered into the method setup initially. If an EDTA sample within the run is more than ± 0.2 from its calculated protein equivalent, a drift correction is performed. The 4 samples preceding and following the corrected EDTA must be recalculated.
United States Department of Agriculture
Food Safety and Inspection Service, Office of Public Health Science
CLG-PRO4.03 Page 5 of 8
Title: Protein Determination by Combustion
Revision: 03 Replaces: CLG-PRO4.02 Effective: 07/27/2009
H. SAFETY INFORMATION AND PRECAUTIONS
1. Required Protective Equipment - Safety glasses, heat-resistant gloves, plastic gloves, laboratory coat.
2. Hazards
Procedure Step Hazard Recommended Safe
Procedures
Unit operates at 220 volts AC and has a high voltage power supply
Crucible combustion tube and reduction tube
Can cause severe burns/electric shock
Extremely hot (700 - 1150
°C)
Turn instrument off and remove metal objects from hands and arms before reaching into the instrument cabinet.
Allow to cool or use suitable tool when they are hot
Pure Oxygen Explosive Remove all ignition sources from the laboratory area
Compressed gas cylinder
Explosive Mount cylinders firmly and have two stage regulators
attached before cylinder valves are opened.
Magnesium
Perchlorate
Strong oxidizer, contact with
flammable materials may cause ignition. Causes irritation to skin, eyes, and respiratory tract
Use in a fume hood.
Sodium Hydroxide Causes burns to all body tissue. Corrosive. Reacts
with some metals to form H2
Use in a fume hood.
3. Disposal Procedures
Procedure Step Recommended Safe Procedures
Copper Dispose in accordance with local, state, and Federal regulations.
Sodium Hydroxide Collect waste in tightly sealed container and store away from non-compatibles in a cool, storage area/cabinet for disposal in accordance with local, state, and Federal regulations.
United States Department of Agriculture
Food Safety and Inspection Service, Office of Public Health Science
CLG-PRO4.03 Page 6 of 8
Title: Protein Determination by Combustion
Revision: 03 Replaces: CLG-PRO4.02 Effective: 07/27/2009
Magnesium Perchlorate Collect waste in tightly sealed container and store away from non-compatibles in a cool, storage area/cabinet for disposal in accordance with local, state, and Federal regulations.
I. QUALITY ASSURANCE PLAN
1. Performance Standard
Analyte Analytical Range % Repeatability Reproducibility
1 < 0.242 < 0.322
Protein
1 Limit may vary due to sample aliquot size and sample type.
2 One Standard Deviation based on historical data.
2. Critical Control Points and Specifications
Record Acceptable Control
Sample must be dried before loading in the
Sample Condition
autoloader sample rack.
Forced Draft Oven 101 ± 1 °C. Sample weight 1.0 ± 0.2 g.
Note: Weigh less sample if % total protein is
out of calibration range.
Reduction reagent No more than 600 assays should be done before re-packing tube.
3. Readiness To Perform a. Familiarization
i. Phase I: Standards- Not Applicable.
ii. Phase II: Fortified samples- Run a set of 5 -10 previously analyzed samples in duplicate. Repeat on two additional days. (Different samples may be used on each day).
iii. Phase III: Check samples for analyst accreditation.
(a) 15 check samples for initial analyst qualification.
(b) Samples submitted by the Quality Assurance Manager (QAM),
United States Department of Agriculture
Food Safety and Inspection Service, Office of Public Health Science
CLG-PRO4.03 Page 7 of 8
Title: Protein Determination by Combustion
Revision: 03 Replaces: CLG-PRO4.02 Effective: 07/27/2009
Accredited Laboratory Program (ALP), or supervisor
(c) Authorization from the Quality Assurance Manager (QAM) and
Supervisor are required to commence official analysis
b. Acceptability criteria.
Refer to I. 1.
4. Intralaboratory Check Samples
a. System, minimum contents.
i. Frequency: 1 per week, per analyst, if samples are analyzed.
ii. Records are maintained by analyst and reviewed by supervisor and laboratory Quality Assurance Manager (QAM).
b. Acceptability criteria.
Refer to section I.1., Performance Standards
If unacceptable values are obtained, then:
i. Stop all official analyses for the analyst. ii. Take corrective action.
5. Sample Acceptability and Stability
a. Matrix: Fresh and processed meat and poultry products. b. Sample receipt size, minimum: 1 lb.
c. Condition upon receipt: Unspoiled and sealed from the air. d. Sample storage
Time and Condition: 24 months frozen or 1 - 3 weeks refrigerated.
6. Sample Set
a. EDTA
b. Meat recovery c. Samples
7. Sensitivity
Method detection limit (MDL): 0.2 %.
J. WORKSHEET
None
United States Department of Agriculture
Food Safety and Inspection Service, Office of Public Health Science
CLG-PRO4.03 Page 8 of 8
Title: Protein Determination by Combustion
Revision: 03 Replaces: CLG-PRO4.02 Effective: 07/27/2009
K. APPROVALS AND AUTHORITIES
1. Approvals on file.
2. Issuing Authority: Director, Laboratory Quality Assurance Division.
4 comments:
Unedited, so kinda long. Sorry, I can edit it later, I'm really sleepy.
September 7, 2011 at 9:10 AM-Karan
September 7, 2011 at 9:10 AMkar, this does not have the source does it?
September 7, 2011 at 10:02 AM-caesey
What?..naa...its in grey..naay ngalan na "source: http://churvabels"
September 8, 2011 at 6:49 AM-Karan
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