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Bulgarian Journal of Agricultural Science, 19 (2) 2013, 197–200Agricultural Academy

ELECTROPHORETIC CHARACTERIZATION OF MILK PROTEINS FROM BULGARIAN RHODOPEAN CATTLE

L. ZAGORCHEV1, M. DIMITROVA1, M. ODJAKOVA1, D. TEOFANOVA2 and P. HRISTOV2*

1 Sofi a University “St. Kliment Ohridski”, Department of Biochemistry, Faculty of Biology, BG – 1164 Sofi a, Bulgaria2 Bulgarian Academy of Sciences, Institute of Biodiversity and Ecosystem Research, BG - 1113 Sofi a, Bulgaria

Abstract

ZAGORCHEV, L., M. DIMITROVA, M. ODJAKOVA, D. TEOFANOVA and P. HRISTOV, 2013. Electrophoretic character-ization of milk proteins from Bulgarian Rhodopean cattle. Bulg. J. Agric. Sci., Supplement 2, 19: 197–200

αs1-casein, κ-casein and β-lactoglobulin show comparatively the highest polymorphism among the bovine milk proteins. Therefore, they have been extensively investigated in respect to identify the correlation between this polymorphism and the qualitative and quantitative milk traits. It is possible, based on the variability of milk proteins, to clarify the phylogenetic relationships between different cattle breeds and to gather the necessary data to preserve the genetic fund and biodiversity of the local breeds. In the present study an optimized 2D electrophoretic method for milk protein profi ling is used to determine the variation in the isoforms of αs1-casein, κ-casein and β-lactoglobulin in the Bulgarian Rhodopean cattle. In addition, the possible relation between this polymorphism and the qualitative features of milk are discussed. Several different genotypes for each of the milk protein genes were established in the investigated population. The method proposed here is a highly sensitive system for milk protein investigation. It could be used to determine the phylogenetic origin of a given cattle breed but also to maintain a highly productive selection and preservation strategy. In addition, the electrophoretic method could be successfully applied to industry in respect of quality control of commercial milk products.

Key words: 2D electrophoresis, β-lactoglobulin, milk proteins polymorphism

*E-mail: peter_hristoff@abv.bg

Introduction

The genetic polymorphism of bovine milk proteins was highly investigated recently as being of great importance in breeding and hybridization strategies as well as studies on the evolution, population structure and preservation of the natural genetic varieties among indigenous breeds. Moreover a num-ber of studies suggest that milk protein polymorphism has a strong infl uence on milk qualitative and quantitative traits and technological properties (Di Stasio and Mariani, 2000; Martin et al., 2002). Such studies were initiated only recently in Bul-garia (Zlatarev et al., 2008; Hristov et al., 2011a, b), a country with a long-term tradition in agriculture and especially cattle breeding. Up to now, most of the breeding strategies were based solely on phenotypic characteristics and information on the genetic structure of the populations is scarce. Recent

interest was focused on the Bulgarian Rhodopean cattle breed (BRC) and its predecessor, the Shorthorn Rhodopean cattle breed (SRC). SRC is one of the two native cow breeds in Bul-garia and one of few that are considered as the last forms of prehistoric European cattle breeds. It was included in the rare breeds and varieties of the Balkans Atlas (Kugler, 2009) with risk status: endangered, with only 197 animals in the popu-lation. Therefore, the investigation on both BRC and SRC should be considered as important scientifi c purpose for Bul-garian, but also for European agriculture in terms of breeding strategies improvement and gene fund preservation.

Bovine milk proteins are subdivided into two classes based on their behavior at pH 4.6 – about 80% caseins (αs1-, αs2-, β- and κ-) that precipitate at this pH values and about 20% whey proteins (mainly β – lactoglobulin and α – lactal-bumin) that remain soluble. Each of the mentioned proteins

198 L. Zagorchev, M. Dimitrova, M. Odjakova, D. Teofanova and P. Hristov

exists in at least two genetic variants, caused by amino acid substitutions in the genes. Our recent studies are focused on the genetic polymorphism of αs1 – and κ – casein and β – lactoglobulin in SRC and BRC as being the most important in terms of milk traits and selection strategies. The present study establishes an electrophoretic method for screening of the above mentioned milk proteins in milk samples.

Material and Methods

Milk samplesMilk samples were collected from the Smolyan area in

Bulgaria from 20 individuals of the BRC breed and pre-served in 0.2 g.kg-1 sodium azide at 4°C. Prior to electro-phoretic separation, all samples were diluted 1000 times and protein concentration was estimated with BCA kit (Pierce) according to producers manual.

Electrophoretic separation Equal amount of protein (15 μg) was applied to each start

on SDS PAGE, 14 % T, 3% C separating gel after incubation

for 3 min. on 100oC in reducing sample buffer. Electrophore-ses were performed according to Laemli (Laemli, 1970). Gels were stained in CBB G250 and caseins and β-lactoglobulin fractions were identifi ed according to standards (Sigma). For 2D-E a denaturing vertical IEF with a fi nal concentration of 6M Urea was performed according to Robertson (Robertson et al., 1987). Carrier ampholytes were Pharmalyte pH 4.2-4.9 and 4.5-5.4 (Sigma) in equal amounts. Prior to second dimension lanes were equilibrated in equilibration buffer and applied to 14% T, 3% C SDS PAGE. Different isoforms of caseins and β-lactoglobulin were identifi ed according to their pI and Mw values.

Results and Discussion

SDS PAGE separation in one dimension showed that β-lactoglobulin isoforms could be successfully discriminated while caseins could not due to the high presence of proteins with similar Mw (Figure 1). Of the 11 offi cially identifi ed LGB allelic forms (Caroli et al., 2009) two (A and B) were found in the BRC population as confi rmed by PCR-RFLP

Fig. 1. 14% T SDS PAGE of β-lactoglobulin (1), αs1-casein (2), κ-casein (3) standards and milk protein samples from Bulgarian Rhodopean Cattle (4-7). Mw of protein electrophoresis standards are shown on the left

199Electrophoretic Characterization of Milk Proteins from Bulgarian Rhodopean Cattle

analysis (Hristov et al., 2012). The two protein isoforms dif-fer in two amino acids substitutes and difference in Mw of about 100 Da and are readily separated on 14% acrylamide gel in both the standard and the milk samples. The apparent Mw was about 15 kDa compared to the expected 18 kDa (Farrell et al., 2004). About 55% of the animals were iden-tifi ed as heterozygous (AB), while homozygous AA or BB animals were respectively about 25 and 20%. The results does not fully confi rm the RFLP profi le that was previously performed (Hristov et al., 2012) but still show the predomi-nance of the A allele.

About half of the milk samples were separated by 2D-E in a narrow pH gradient (pH 4.2 – 5.4) and 14% T SDS PAGE. Protein identifi cation was based on the expected pI and Mw

values (Farrell et al., 2004) and the general 2D bovine milk protein profi le that was previously established (Holland et al., 2012). The results for β-lactoglobulin were further con-fi rmed though two to fi ve different spots with differing pI were observed in some of the samples. We attributed the ex-istence of the lower abundant β-lactoglobulin spots to pos-sible chemical modifi cations caused by the preservation pro-cedure or the prolonged storage as this protein was shown to be susceptible to such changes during a variety of treatments (Meltretter et al., 2008).

Two κ-casein isoforms were identifi ed, differing in pI (Figure 2). The discrimination between the two isoforms was problematic due to the contradictory data about their exact pI (Farrell et al., 2004) and the low abundance of the κ-casein

Fig. 2. 2D-E separation of milk proteins from Bulgarian Rhodopean Cattle. Mw of protein electrophoresis standards are shown on the left. β-lactoglobulin (β-lac), A and B isoforms, αs1-casein (αs1-cas) and κ-casein (κ-cas) fractions are

shown

200 L. Zagorchev, M. Dimitrova, M. Odjakova, D. Teofanova and P. Hristov

spots in all samples. The latter should be attributed to the mi-celle formation, shown for κ-casein that generally impedes the successful separation (Hidalgo et al., 2010). As for the αs1-casein a number of spots were observed that correspond to the pI and Mw expected (Figure 2). Extensive phosphory-lation (8 to 9 phosphorylation sites) (Holland et al., 2012) and alternative splicing of the CSN1S1 mRNA (Giambra et al., 2010) have been proved for this milk protein and there-fore lead to a variety of protein spots as separated by 2D-E. Nevertheless at least two predominant isoforms could be distinguished and all of the individuals investigated were proposed to be BC heterozygous animals. Previous analyses showed that about 70% of the population was heterozygous (Hristov et al., 2012).

Conclusion

The proposed 2D-E method for milk protein character-ization proved to be a fast and reliable procedure for at least some of the milk proteins like β-lactoglobulin. Although not conclusive for determination of the genetic polymorphism, it could be very useful in studying the structure of different cattle breed populations and for evaluation of raw milk qual-ity, health condition of particular lactating individuals but also for screening of commercial milk products for possible additives like soybean.

AcknowledgmentsThis study was supported by grant YRG 02/23 28.12.2009

from the National Science Fund of the Bulgarian Ministry of Education, Youth and Science, Sofi a, Bulgaria.

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