Analytica Chimica Acta 393 (1999) 227±234

Determination of protein and casein in milk by fourth

derivative UV spectrophotometry
Qiaoqian LuÈthi-Peng*, Zdenko Puhan
Laboratory of Dairy Science, Institute of Food Science, Swiss Federal Institute of Technology, (ETH), CH-8092 ZuÈrich, Switzerland

Received 26 August 1998; received in revised form 15 October 1998; accepted 8 November 1998

Abstract

The aim was to develop a simple and rapid method for determination of milk protein in general, and casein content in
particular, in milk without involving preparation procedures such as skimming and casein precipitation. Fresh raw milk
samples were diluted with 6 M guanidine-HCl buffer before spectroscopic scanning. The total protein and casein contents of
45 milk samples determined by this method were not statistically signi®cantly different from the values obtained by the
reference method based on Kjeldahl determination of N (p < 0.05). By using fourth derivative UV spectrophotometers, the
determination of total protein and casein content in milk can be achieved directly in a single run. Treatments of milk such as
heating, homogenization, preservation or hydrolysis with protease had no in¯uence on the accuracy of protein determination
in milk by this method. The accuracy of determination with 0.03% total protein and 0.04% casein for the reference method
could not be achieved under the condition used, due to the effect of high dilution rate. # 1999 Elsevier Science B.V. All rights
reserved.

Keywords: Milk protein and casein determination; Fourth-order derivative spectroscopy

1. Introduction replaced by rapid methods such as infrared (IR)

Rapid and direct determination of casein content in
milk has long been a wish for breeding organizations,
the dairy industry as well as for control laboratories.
The most widely used method for measuring the
casein content is still that of Rowland [1], a method
that involves precipitation of casein at pH 4.6, deter-
mination of nitrogen in milk and whey by Kjeldahl and
calculation of casein as the difference between total
and whey protein. As the procedure is laborious and
time-consuming, this method has been partially
*Corresponding author. Tel.: +411-632-5372; fax: +411-632-
1156; e-mail: luethi@ilw.agrl.ethz.ch
spectroscopy and the dye-binding method [2,3]. How-
ever, in both cases, precipitation of casein is still
required and corrections due to the change in milk
matrices caused by precipitation are necessary.
Luf and Brandl [4,5] introduced the use of second
derivative spectrophotometry for determining the heat
classi®cation of skimmed liquid and dried milk based
on the amount of undenatured whey protein remaining
after heat treatment in the pH 4.6 ®ltrate. The method
is based on the UV absorption of trptophan (Trp) and
tyrosine (Tyr), each with characteristic maxima and
minima between 270±300 nm. An optimization of a
second derivative absorption spectrophotometric
method for quantifying milk protein fractions of case-

0003-2670/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 0 0 3 - 2 6 7 0 ( 9 8 ) 0 0 8 2 3 - X
228 Q. LuÈthi-Peng, Z. Puhan / Analytica Chimica Acta 393 (1999) 227±234
inates and milk powders has been down by De Jong
and Olieman [6]. Meisel [7] has applied fourth deri-
vative UV spectrophotometry for rapid determination
of the casein-to-whey ratio in milk or milk products,
without necessarily knowing the total protein content.
The aim of this study was to measure directly the
milk protein fractions by fourth derivative UV-spec-
trophotometry without procedures such as skimming
of milk and casein precipitation. The impact of heat-
ing, homogenization, preservation or hydrolysis of
milk proteins on the protein determination was also
considered.
2. Materials and methods
2.1. Materials
N-acetyl-L-tryptophan ethyl ester, N-acetyl-L-tyro-
sine ethyl ester and bovine b-lactoglobulin (b-Lg)
were Sigma products. Bovine a-lactalbumin (a-La),
trypsin and thermolysin were purchased from Fluka.
Hammarsten casein was obtained from Merck.
2.2. Samples
Twenty-eight samples of fresh milk from individual
cows and buffaloes, and 17 fresh bulk cow milk
samples were collected during the period from March
to October 1997 from two farms in Zurich and
Emmental, Switzerland.
2.3. Reference analysis
As reference, the International Dairy Federation
(IDF) methods for total nitrogen in milk [8] and for
casein content [9] were used. The total protein content
throughout the work is expressed in g/100 g milk and
derived from the difference between total N and non-
protein-N [1].
2.4. Fourth derivative spectrophotometric analysis
Prior to the measurements, samples were brought to
408C in a water bath, and cream was resuspended by
gentle swirling and inversion of the sample container.
20 ml of milk was diluted 50 times with 6 M guani-
dine-HCl (Gdn) in 0.1 M Na acetate buffer, pH 5.0,
and vortexed. A 10 mm quartz cuvette was used, and
UV spectra were recorded against the Gdn buffer on a
Cary-1 UV spectrophotometer (Varian Analytical
Instruments) at ambient temperature using a double-
beam from 320 to 260 nm at a scan rate of
100 nm minÿ1, average time 0.3 s and interval
0.5 nm. To compute the fourth derivative spectra,
the software package GRAMS/32 (Galactic Indus-
tries) was used. The fourth derivative spectra were
calculated from UVabsorption spectra by means of the
Savitsky-Golay algorithm [10] using a quadratic poly-
nomial ®t over 21 points. The amplitudes of the
signature peaks and troughs were obtained directly
from the fourth derivative spectra. Each sample was
analysed at least in duplicate.
2.5. Treatment of raw milk samples for fourth
derivative spectrophotometry
To ascertain to what extent the treatments used in
milk processing affect the fourth derivative spectra,
the following was carried out before the milk sample
was scanned.
2.5.1. Heating
Samples were heated up to 958C in test tubes
(20 ml, covered) using a water bath, with holding
times of 3±10 min at the desired temperature.
2.5.2. Homogenization
The raw milk sample was either homogenized by a
Polytron PT1035 for 5±10 s or by a built-in homo-
genizer, Milkoscan 4000 (Foss Electric, Denmark) at
180 bar at 408C.
2.5.3. Preservation
Raw milk was preserved with a ®nal concentration
of 0.02% Bronopol; or 0.15% H2O2; or 0.2% Na azide
and stored at ambient temperature for one day.
2.5.4. Proteolysis
Skimmed milk was incubated directly with trypsin
(1 mg mlÿ1) or thermolysin (1 mg mlÿ1) at ambient
temperature for 1 h.
2.6. Calibration and quantification
The calibration for casein (Cn) and whey protein
(WP) content was performed using a mixture of
 Q. LuÈthi-Peng, Z. Puhan / Analytica Chimica Acta 393 (1999) 227±234
Hammarsten casein with puri®ed whey protein con-
taining b-Lg and a-La (b-Lg : a-La ˆ 2.5 : 1). The
casein content of Hammarsten casein was calculated
by its nitrogen content multiplied by 6.38, the nitro-
gen-to-protein conversion factor. The concentrations
of b-Lg and a-La were determined spectrophotome-
trically using extinction coef®cient (absorptivities) of
0.96 (mg/ml)ÿ1 cmÿ1 at 278 nm [11], and 2.01 (mg/
ml)ÿ1 cmÿ1 at 280 nm [12]. The whey protein content
of the mixtures ranged from 0 to 100%. Calibration for
total protein (TP) was generated from a mixture
containing 80% of Hammarsten casein and 20% of
whey protein (b-Lg : a-Laˆ2.5 : 1) and the concen-
tration was set from 0.1 to 1.2 mg protein mlÿ1. The
effect of the remaining milk components on both
calibration lines was veri®ed using a milk sample
with a protein composition previously determined
by the reference method, and subsequently enriched
with Hammarsten casein or whey proteins (b-Lg :
a-La ˆ 2.5 : 1) to give whey protein contents from
10 to 50%.
3. Results and discussion
3.1. Principle of the method
The characteristic protein absorption band at ca.
280 nm arises from the combined oscillator strengths
of the lowest energy ±* transitions of the three
aromatic amino acids Trp, Tyr and phenylalanine
(Phe). The molar absorptivity for Phe is quite low
in this region, thus can normally be ignored [13]. Due
to the lower molecular symmetry of Trp and Tyr, the
molar absorptivities of Trp and Tyr are much higher
than that of Phe, being 5.5 and 1.34 mMÿ1 cmÿ1 at
their respective maxima of 278 and 275 nm [14]. They
are the main contributors to the protein absorption
spectrum. Separating the characteristic contribution of
these two amino acids can be achieved by differentia-
tion of the absorption spectrum. The maximum of a
parent spectrum in derivatives of odd order corre-
sponds to a passage through zero, and in even-order
derivatives it corresponds to an extreme value, either a
minimum or maximum. A derivative of even order is
generally used, since in this case the extreme values
correspond to one another. With increasing order of
derivatisation the sharpness of the bands increases,
229
while the band width decreases. Thus the resolution is
improved signi®cantly. A higher-order derivative
improves the resolution of complex spectra at the cost
of decreasing the signal/noise ratio. The fourth deri-
vative has been considered the best compromise
between signal/noise ratio and resolving power [15].
Since derivative absorption spectrophotometry fol-
lows the Beer-Lambert law [16], the amplitude of
any peak and trough given in the fourth derivative
spectrum is linearly related to the chromophore con-
tent.
In general, spectral changes can result from incor-
poration of an amino acid into a polypeptide, and these
effects can be eliminated by using a denaturant,
namely urea or guanidine-HCl, to such an extent that
the analogs of Trp and Tyr closely mimic the spectra of
their corresponding residues within the denatured
protein, and all chromophores are normalized to sol-
vent by Gdn [18]. Fig. 1 shows the near-UVabsorption
and the fourth derivative spectra of Try and Tyr
analogs at 1 mM concentration and of a raw milk
sample before and after it was skimmed. All samples
were prepared in 6 M Gdn buffer. From the fourth
derivative spectra in Fig. 1 it can be seen that with the
analog compounds, N-acetyltryptophan ethyl ester
and N-acetyltyrosine ethyl ester, the trough at
294 nm for Trp had the least overlap with the Tyr
signal. This feature has been successfully used to
quantify Trp and Tyr residues in proteins [15], as well
as the whey protein content in total protein [7], based
on the different Trp and Tyr contents in whey proteins
and casein. The derivative UV-spectrum has also the
advantage of being largely unaffected by constant
background caused by either other UV absorbing
substances in the sample [17] or turbidity [19],
because derivatives eliminate the linear background
function. The turbidity caused by milk fat had l
ittle contribution to the fourth derivative spectrum
(Fig. 1).
The fourth derivative spectra of a series of casein
and whey protein (b-Lg : a-La ˆ 2.5 : 1) model mix-
tures are shown in Fig. 2. In these mixtures, the whey
protein content varied in the range 0 to 100%, whereas
the total protein content of the mixtures was kept
constant at 1.1 mg protein mlÿ1. The amplitudes of
the peaks at 283.5 and 291 nm, and of the troughs at
287 nm are due to the absorption by both Tyr and Trp,
and the trough at 294.5 nm is speci®c for Trp. The
230 Q. LuÈthi-Peng, Z. Puhan / Analytica Chimica Acta 393 (1999) 227±234
Fig. 1. UV-absorption (A) and fourth derivative (B) spectra of a raw milk sample before (RM) and after (SM) it was skimmed. Total protein
2.907%, casein 2.427%, casein number 83.49. The concentration of N-acetyl-L-tryptophan ethyl ester (Trp) or N-acetyl-L-tyrosine ethyl ester
(Tyr) was 1 mM.
Fig. 2. The fourth derivative spectra of mixtures containing casein
and whey proteins (b-Lg : a-La ˆ 2.5 : 1). Whey protein content,
0±100% as indicated; total protein, 1.1 mg mlÿ1.
amplitudes of the troughs at 287 and 294.5 nm, as well
as the amplitude of the 291 nm peak increased pro-
portionally with increasing whey protein content in
the mixture, while the amplitude of the peak at
283.5 nm remained unchanged, because of the con-
stant total protein content in the mixtures. Thus, it can
be concluded that the contribution of casein and whey
proteins at 283.5 nm is identical, which has been
proved by calibration using only casein or whey
protein (b-Lg : a-La ˆ 2.5 : 1). Therefore, the ampli-
tude of the peak at 283.5 nm was used to quantify the
total protein content. In the range 0.1±1.1 mg
protein mlÿ1, the absolute ratio of the amplitude at
the 294.5 nm trough to that at the 283.5 nm peak
remained the same, i.e. 0.25 for casein and 0.66 for
whey protein (b-Lg : a-La ˆ 2.5 : 1), the reason being
that at 294.5 nm Tyr has the least contribution to the
fourth derivative value and the Trp content in whey
protein is more than twice more than that in casein.
This makes it possible to use the absolute value of the
294.5 nm trough over the peak at 283.5 nm to quantify
the whey protein and casein content, as reported by
Meisel [7]. Total protein and casein content of a milk
sample can be obtained in a single derivative spectro-
scopic measurement (Eqs. (1) and (2)).
TP…g=100 g† ˆ 380‰4 A=4283:5 Šfdilution (1)
Cn…g=100 g† ˆ TP…1 ÿ WP=TP† (2)
 Q. LuÈthi-Peng, Z. Puhan / Analytica Chimica Acta 393 (1999) 227±234 231

Fig. 3. Linear correlation of 4 A=4283:5 to total protein (TP) in graph A, and of 4 A=4294:5 =4 A=4283:5 to whey protein in total protein
(W/TP) in graph B. Key to symbols: Mixtures containing casein and purified whey protein (b-Lg : a-La ˆ 2.5 : 1), with which the calibration
lines were constructed (&). A raw milk sample with known protein composition enriched with casein (*), or with whey protein (b-Lg : a-
La ˆ 2.5 : 1) (~).

where 3.2. Casein content of raw milk samples

j4 A=4294:5 j To ascertain as to whether the fourth derivative

WP=TP ˆ 2:5 ÿ 0:6
j4 A=4283:5 j
It is obvious that determination of TP content is also a
function of dilution factor, while this factor plays no
role in determination of whey protein content in total
protein (WP/TP). Because these equations were cali-
brated by the solutions containing only milk proteins,
the effect of other milk components was veri®ed as
described in the experimental section. The results
indicate that the remaining milk components had no
effect on the calibration lines (Fig. 3).
method might be an alternative for direct determina-
tion of casein in raw milk, a comparison was made
between casein contents measured by this method and
by the reference method. The range of total protein,
casein content and casein number for 34 fresh cow's
milk samples determined are listed in Table 1. These
results were evaluated in terms of mean difference,
slope, intercept, correlation coef®cient and standard
errors associated with each parameter (Table 2). The
statistical results revealed a linear correlation between
the two methods. When calculating the slope and

Table 1
Milk protein and casein determinations by reference method (Ref) and derivative method (DS)

Milk samples n Method Total protein mean Casein mean Casein number a
(range) % (g/100 g) (range) % (g/100 g) Mean (range)

Cow's milk bulk 17 Ref 3.099 (2.863±3.478) 2.617 (2.388±2.944) 84.47 (82.62±87.30)
DS 3.080 (2.660±3.420) 2.590 (2.245±2.923) 84.35 (82.37±86.11)

Cow's milk individual 17 Ref 3.215 (2.450±4.200) 2.664 (1.922±2.459) 81.86 (77.97±85.15)
DS 3.204 (2.420±4.370) 2.690 (1.950±3.624) 83.07 (79.01±86.68)

Cow's milk total 34 Ref 3.173 (2.450±4.200) 2.640 (1.922±3.338) 83.28 (77.97±87.30)
DS 3.150 (2.420±4.370) 2.640 (1.950±3.584) 83.47 (79.01±87.30)
a
Casein numberˆcasein/total protein  100.
232 Q. LuÈthi-Peng, Z. Puhan / Analytica Chimica Acta 393 (1999) 227±234

Table 2
Statistical descriptors for milk total protein and casein content determined by fourth derivative spectrophotometry

Sample n MD SDD Slope a y Intercept a ra SE of DS value CV

Cow's milk
total protein 34 0.018 0.064 1.065 ÿ0.245 0.9686 0.064 1.03
casein 34 0.001 0.068 1.073 ÿ0.195 0.9534 0.056 1.04

Buffalo milk
total protein 11 ÿ0.016 0.073 1.007 0.032 0.9624 0.150 1.6
casein 11 ÿ0.094 0.089 1.019 0.020 0.9358 0.225 2.8
Abbreviations: n, number of milk samples analyzed; MD, mean difference from the reference values; SDD, standard deviation of differences
from the reference values; r, correlation coefficient; SE, standard error; CV, coefficient of variation.
a
Regression analysis used the reference values as the independent (x) variable and the DS values as the dependent (y) variable.

intercept of derivative value versus reference value, no not the milk proteins are denatured or degraded during
statistically signi®cant differences were found from processing prior to derivative spectrophotometry. This
the theoretical values at p ˆ 0.05. Repeatability, Sr, has been proved in the investigations concerning the
de®ned as the standard deviation of the differences impact of denaturation of whey protein by heating and
between duplicates was 0.096% for total protein and enzymatic degradation of milk protein. In addition, no
0.112% for casein. The values for an additionally difference was found when analyzing the protein
measured unknown sample of cow's milk at the fractions of a milk before and after it was fermented
95% con®dence interval were 0.064% and to yoghourt and stored at 48C for one week. Therefore,
0.056% for total protein and casein, respectively, this method can also be used to measure the whey
with fourth derivative spectrophotometry, and protein-to-casein ratio in pasteurized or UHT milk,
0.066% and 0.054% with the reference method, milk powders or milk protein preparations.
respectively.
Table 3
3.3. Effect of treatment of milk on protein Influence of treatment on milk protein determination by fourth
determination derivative spectrophotometry (Changes expressed in % of the
measured value of the milk before treatment. Experimental error:
2.41% for TP and 3.85% for casein)
The effect of heating, homogenization, storage,
preservation and proteolysis of milk proteins applied Treatment Total protein Casein
in milk processing on protein determination by deri- Heating 958C, 10 min 0 0.08
vative spectrophotometry is shown in Table 3. Within
experimental error, heating up to 958C for 10 min, Homogenization
homogenization, preservation with Bronopol, H2O2 or Polytron, 10 s 0 0.33
Na azide at the concentration commonly used in milk 180 bar 0 0.2
trading, hydrolysis and storage at 48C for up to two Preservation
days had no signi®cant effect on the derivative spectra 0.02% Bronopol 0 1.12
of the milk proteins. The milk samples which had been 0.15% H2O2 1.16 0.32
incubated with trypsin or thermolysin showed no 0.2% Na azide 0 0.28
bands in urea-polyacrylamide gel electrophoresis,
Storage at 48C for
indicating that the milk protein fractions had been 24 h 0 0.14
extensively hydrolysed. 48 h 0 0.49
The accuracy of the method is due to the unfolding
of proteins and polypeptides in Gdn solution, leading Proteolysis with
trypsin 0.98 0.11
to a uniform UV absorption of the chromophores Tyr
thermolysin 0.91 0.41
and Trp. Therefore, it makes no difference whether or
 Q. LuÈthi-Peng, Z. Puhan / Analytica Chimica Acta 393 (1999) 227±234
3.4. Source of potential errors
The model milk with which the calibration plots
were generated contained Hammarsten casein and a
mixture of puri®ed whey protein containing b-Lg and
a-la (b-Lg : a-La ˆ 2.5 : 1), which assumes the milk
protein composition remains the same, though the
composition varies with breed, stage of lactation
and season. Less variation is expected for the absolute
ratio of amplitude at trough 294.5 nm to that at peak
283.5 nm of the derivative spectrum for casein than for
whey protein. This ratio is determined by the ratio of
Trp/Tyr in milk proteins. Casein has an average Trp/
Tyr ratio of 0.187 which is very close to as1-CN.
Although the Trp/Tyr ratio of b-CN is 0.25, the Trp
and Tyr contents of b-CN are at least a factor of 2
smaller. This is also true for as2-CN and k-CN. This
would mean that the Trp/Tyr ratio of casein without
b-CN or k-CN is just slightly different, being 0.179
or 0.198, respectively. Milk clotting enzyme splits
k-CN into para-casein and casein macropeptide
(CMP); the latter contains neither Trp nor Tyr.
Although the b-CN degradation products, g-CNs,
contain a Tyr residue, this type of degradation takes
place only to a very limited extent that the difference
in Trp/Tyr ratio caused by losing g-CNs is negligible.
Unlike casein, the difference in contribution to the
derivative spectrum by individual whey protien is
more apparent. The ratio of Trp/Tyr in major whey
Fig. 4. Linear dependence of total protein and casein values obtained by derivative spectrophotometry (DS) with those obtained by the
reference method (Ref). (*) bulk cow's milk, (&) individual cow's milk, (~) buffalo milk.
233
proteins, b-Lg and a-La, is generally much higher
than that in casein, being 0.5 for b-Lg and 1.0 for a-
La. This ratio is, however, lower (0.1) in bovine serum
albumin (BSA). Increasing of BSA by 2%, a decrease
of about 2% in whey protein content was obtained,
while no effect on TP determination was observed.
Such a negative response of BSA was also noticed by
De Jong [6].
The crucial role of speci®c calibration in this
method has been shown in comparing the data on
individual and bulk cow's milk samples and individual
buffalo milk samples. The average protein composi-
tion of buffalo milk is similar to that of cow's milk
[20], though the protein content is much higher in the
former. Larger differences between the values
obtained by both the derivative spectrophotometric
and reference methods were observed for individual
cow and buffalo milks compared to bulk cow's milk
(Fig. 4). This result suggests that the more the milk
protein composition deviates from the model milk, the
larger is the difference between the values obtained by
the two methods, because the model milk consisted of
cow's milk proteins and its composition corresponded
to the average value for cow's milk. Nevertheless, a
linear correlation could also be found for both the
casein content and total protein of buffalo milk sam-
ples determined by the derivative spectrophotometric
and reference methods (Table 2). Data on buffalo milk
samples also showed that up to 10% of milk fat in the
234 Q. LuÈthi-Peng, Z. Puhan / Analytica Chimica Acta 393 (1999) 227±234
sample had no signi®cant effect on protein determina-
tion under the conditions used.
The maximum difference of 0.03% total protein and
0.04% casein between duplicates of the reference
method could not be achieved by derivative spectro-
photometry as described in this study. This is due to
the effect of the high dilution. For 50 times dilution,
the difference of 0.003% total protein between two
spectrophotometric determinations will be increased
50 times in expressing the original protein concentra-
tion of the sample. As a matter of fact, the milk protein
fractions could be determined by using a 2 mm cuvette
with 10 times dilution as accurately as by using a
10 mm cuvette with 50 times dilution. That is to say,
the precision can be improved signi®cantly by
decreasing the light path length.
The method described is based on the UV absorp-
tion of aromatic amino acids of the denatured protein,
resulting in selective protein determination. The
advantage of using guanidine-HCl as denaturant
was not only that it uni®ed the environment of the
chromophores but also that it reduced the turbidity
caused by milk fat. It is presumable that Gdn acts as an
emulsi®er to reduce the size of fat globules. A 50-fold
diluted whole cow's milk increases its OD280 (absor-
bance at 280 nm) in the presence of 6 M Gdn-HCl
from 0.6 to 1.0, in 7.2 M urea from 0.6 to 2.8 and in
water from 0.6 to 3.8. For most spectrometers, the
Lambert-Beer law is obeyed within an absorbance
value of 2.0, although some companies guarantee that
the law is obeyed within an absorbance of 4.0. It is
interesting to note that for the same milk sample,
OD280 measured in a 2 mm cuvette with 10 times
dilution was 27% higher than that measured in a
10 mm cuvette with 50 times dilution. This indicates
that the presumed emulsifying effect of Gdn depends
upon the ratio of Gdn to milk fat, though it had no
effect on protein determination.
It can be concluded that fourth derivative absorption
spectrophotometry provides a simple method for
direct determination of total protein, and especially
of the casein to whey protein ratio in liquid milk, as far
as speed and simplicity are concerned. It may also be
applicable to milk powder and to dairy products
containing hydrolysed milk proteins, such as baby
milk powder or fresh cheese. In such cases a speci®c
calibration is required.
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