Application of derivatives of UV spectra and similarity indices to

Application of derivatives of UV spectra and similarity indices to

POLIMERY
106
2003 , 48, nr 2
P]OTR MINKIEWICZ, IERZY DZIUBA-), DoIąoTA NAŁĘCZ
University of Warmia and Mazury in Olsztyn, Chair of Food Biochemistry
Plac Cieszyriski 1, 70-726 Olsztvn-Kortowo, Poland
Application of derivatives of UV spectra and similarity indices to
identification of milk proteins separated ĘPi"g reversed-phase
high performance liquid chromatography '
Summary
The method which may be applied as a tool for ider.rtification of
proteins separated using reversed-phase high performance liquid chromatography, basecl on similaritv indices of UV spectra derivatives, was proposed.
Commerciai milk protcin preparations such as sodium and calcium cascinates, rvhey protein concentrate alrd milk protein coprecipitate as tvcll as
standards of milk proteins were uscd in the expcriment. Statistical signifi
cance of difference between first alrd secorrd similarity indeX, evaluatcd łviŁi-l
Student's, test lvas used as a criterion to choose the mathematical trcatmcnt
of spectra as weil as to prcclict reliability of identification. First dcrivatives of
spectra revealed thc highcst value of f paramcter. The presence of statisticailrr
significant difference between first and second similarity indices always lcd k)
correct idcntification of proteins. The presented method er.rabled appropriatc
identification of caseins in commercial milk protein preparations, but failcd in
the case of whcv proteir.rs.
Key words: identification of proteins, reversed-phase HPLC, UV spectroscopy, similarity ir.rdices of UV spectra deri\ratives.
Although mass spectrometry is the most cfficient
method applied for identification of proteins and pcptides separated using reversed-phase irigh performancc
liquid chromatography (RP-HPLC) [1], in many cases
identificatior.r may be performed using relativelv simple
photodiode-array (PDA) deteciors. Such detectors arc
commonly used [2].
High pcrformance liquid chromatography on-line
with UV spectroscopy \'vas applied in numerous experiments concerning identification of proteins and/or pcptides [3-13]. In some cases this technique was used as
supplementary one for mass spectrometry [9, 11, 13] or
electrophoretic methods [10].
Calculation of spectra derivatiyes in order to enhance
resolution is commonly used procedure for data interpretation concerlirlg peptides and proteins. This procedure
was applied in most of experiments concernir.rg UV spcctra-based idcntification of peptides and/or proteins separated by HPLC 13, 4, 6-131. On the other hand common
procedure for identification of any substances scparated
')
To rvhom all corcsponclcnce sirould bc addrcsscct, c mail: ctz' ba@
uwm.cdu.p)
'')
A PaPcr scnt io Editorial officc bctlĄ,ccll rcscarch rvolks b bc PubIishcd jn thc sPccia] issue of "PoIimcry" dcvoŁPd to biopo]ymcrs (NC)
1.
/2003).
with HPLC is in comparison with standarcl bv r.ncans of
calculation of the similarity indices of spectla. Similariq
indcx is defined as cosinus of angle bciwccn \rcctors jn
multidimcnsional space (named match anglc) bcing
mathematical rcpresentation of spcctra. Thc calculation
of nratch angles betłveen raw spcctr'a lras trocrr itrtrrl
duced as a tool for qualitative analvsis of prcpticlcs [5, 9].
The aim of Plesent study \Ąlas to PIoP()se a StratcgY
inciuding both calculatior.r of dcrivaLivcs of UV spcctra
and similarity indices for iclcntificatior.r of milk protcirrs
separated urith rcversed-phasc high pcrformance liqrucl
chromatograplry. The second goal \,vas to find a critcriorr
to evaluate a method performancc.
EXPERIMENTAL
Materials
Standards of milk protcins: B-casein (13 Cn), r-ca:rcin
(r-Crr), nlactalbumin (cr-La), llJactoglobrrlin (lJ-Lg) an.t
bovine serum albumin (BSA) have L.reen purchasccl flonr
Sigma. As regards orl-casein (o.t-Cu) ancl u.:-cascin
(ols:-Cr.r) they were obtaincd usir.rg anion-cxchansc chro-
natography [14] from whole casein precipjtatcd lrorr
the skim milk of individual Jersey cor'vs. The commcr ci.r
milk protein preparations; sodium cascinatc, calciunr
I
\ń.yPi( .i'romnłairłnd milkPfuEinG
pte.ipible lrav. b&ł Pll.hasg] froń lh. L1cPol 0lo
cmcinaiq
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The
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