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Isolation, Purification, and Identification of Protein Associated with Corn Fiber Gum Madhav P. Yadav,* Alberto Nu~nez, and Kevin B. Hicks Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038, United States ABSTRACT: Corn fiber gum (CFG), an alkaline hydrogen peroxide extract of the corn kernel milling byproduct “corn fiber”, is a proteinaceous arabinoxylan with protein content ranging from ca. 2 to 9% by weight for CFG samples isolated from different corn milling fiber sources. Several studies have suggested that protein associated with CFG could be partly responsible for its excellent emulsifying properties in oil-in-water emulsion systems. Nevertheless, the composition and identity of the protein component has never been determined. In the present study, CFG was deglycosylated by treating with trifluoromethanesulfonic acid, and the resulting proteins were purified by passage through C18 solid phase extraction cartridges. The proteins were then separated and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein band from the gel was treated with a proteolytic enzyme, chymotrypsin, and the resulting peptides were cleaned using C18 Zip Tip pipet tips and analyzed using matrixassisted laser desorption/ionization with automated tandem time-of-flight mass spectrometry. The partial sequences derived from the mass spectrometry analyses of the resulting chymotryptic peptides were found to be similar to the 22-kDa alpha-zein Z1 (az22z1) protein (a major storage protein in corn endosperm) when queried against the primary sequences from the National Center for Biotechnology Information database. This is the first report that this hydrophobic protein is associated with CFG and may explain why CFG is an excellent emulsifier for oil-in-water emulsion systems. KEYWORDS: corn fiber, arabinoxylan, deglycosylation, zein, MALDI-TOF/TOF, mass spectroscopy
’ INTRODUCTION Corn fiber gum (CFG) is an arabinoxylan (hemicellulose B) isolated from deoiled and destarched corn fiber (a low value byproduct of corn wet milling) by an alkaline hydrogen peroxide extraction process.1 The wet/dry milling processing of corn grain produces several product streams, e.g., starch, protein (gluten meal), gluten feed, germ, oil, germ meal (spent flake), steep liquor, and corn fiber (from endosperm and bran). All fuel ethanol facilities using corn grain ferment the starch fraction and recover and sell the other streams as coproducts to offset the cost of fuel ethanol production. In recent years, research into finding more valuable products from corn milling coproducts has intensified due to the significant increase in the production of fuel ethanol from corn. Development of new and higher-value commercial products from low-valued coproducts could help to significantly reduce the cost of fuel ethanol. Corn fiber, which is produced in large amounts from corn milling industries in the USA, is mostly used as an ingredient in low protein animal feed products and sold for a few cents per pound. However, it could be used as starting material for producing high value CFG in about 30% overall yield. CFG dissolves well in water, making a low viscous solution and so it can be used as a bulking, bodying, emulsion stabilizing, and protective colloid agent in which low-viscosity, high-solid gum solutions are required. It has several useful properties, e.g., adhesive, thickening and stabilizing,2 and film forming and emulsifying.1,3 5 The structural investigations have shown that CFG consists of a highly branched xylan backbone with side chains containing arabinose, galactose, glucose, and glucuronic acid,6 and a small percentage (1 5% depending upon the fiber source) of protein.1,7 It is well known that gum arabic has excellent emulsifying properties for beverage emulsion This article not subject to U.S. Copyright. Published 2011 by the American Chemical Society
systems and that its protein components play an important role in its emulsification properties.8,9 It is widely used in the soft drink industry10 to stabilize flavor oil emulsions in water. The emulsifying properties of this naturally occurring emulsifier (gum arabic) decreases on reducing or removing its minor protein component.8,11 Dickinson11 also explained that the carbohydrate structure of hydrocolloid gums does not contain any significant proportion of hydrophobic groups, and therefore, the emulsion stabilizing capability of polysaccharides is dependent upon the hydrophobicity of the attached protein. The above explanation was strongly supported by Brummer, et al.,12 who demonstrated that the purified fenugreek gum containing 0.6% residual proteins had a lower surface activity than the higher protein-containing original unpurified gum. Our previous studies have clearly indicated that CFG is an excellent emulsifier and can be a potential gum arabic replacer for oil-in-water emulsion systems.1 As in gum arabic, our previous studies indicate that its protein component plays an important role in its emulsifying properties.1,7,13 The objective of the present study was to isolate, purify, and identify the protein associated with CFG to fully understand its role in functional properties.
’ MATERIALS AND METHODS Materials. The oven-dried “fine” corn fiber (fiber originating from the endosperm portion of corn kernels) sample was kindly provided by ADM Research. It was ground to a 20-mesh particle size using a Wiley Received: September 1, 2011 Accepted: October 31, 2011 Revised: October 27, 2011 Published: October 31, 2011 13289
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mill and deoiled by extracting with hexane.14 Starch was removed by heat-stable Termamyl α-amylase (Novozymes, Inc., Davis, CA) treatment.15 Trifluoromethanesulfonic acid (TFMS) was purchased from Sigma Chemical Company. Corn zein was obtained from Showa Sangyo Co. Ltd., Tokyo, Japan, distributed by Chugai Boyeki (America) Corp., New York. Isolation of Corn Fiber Gum. CFGs were extracted from deoiled and destarched corn fiber according to the alkaline hydrogen peroxide procedures of Yadav et al.13 with some modification. Protein Analysis. Protein (N 6.25) was determined according to AACC Approved Method 46-30.16
Deglycosylation of Corn Fiber Gum Using Trifluoromethanesulfonic Acid (TFMS). CFG was dried in a vacuum oven
at about 50 °C for overnight. All subsequent steps were performed at 0 °C or below. Anhydrous toluene (10%) was added to TFMS in a glass vial with a Teflon lined cap to make a 90% TFMS solution. The solution was mixed and immersed in a dry ice/ethanol bath and allowed to cool for a few minutes. A CFG sample (20 mg) was weighed into a screwcapped glass vial with a Teflon lined cap, and 0.5 mL of TFMS/toluene mixture (90/10) was added very slowly by using a chemical resistant and dry (water-free) syringe. The solution was mixed gently and kept in the dry ice/ethanol bath ( 20 °C or below). The vial was shaken gently after 5 min to dissolve the sample completely. After another 5 min, the vial was shaken one more time and incubated in the dry ice/ethanol bath for 4 more hours with gentle shaking after every hour. After each shaking, the vial was opened briefly to release vapor. The reaction mixture was then neutralized by a gradual addition of 1 mL of 3:1:1 pyridine/ methanol/water (previously cooled on dry ice/ethanol bath) and was then incubated in the dry ice/ethanol bath for 5 min followed by incubation in a wet ice bath at 0 °C for 15 min. The reaction mixture was then further neutralized to pH 5 6 with 2% ammonium bicarbonate. The neutralized reaction mixture (0.5 mL) was then applied to a C18 solid phase extraction cartridge (Michrom Bioresources, Inc., Auburn, CA) (previously washed with acetonitrile/water, 1:1, TFA 0.1%). The filtrate from the cartridge was collected, and the cartridge was further washed with water (0.5 mL, 2). The protein, adsorbed by the stationary phase in the cartridge, was eluted with 100 μL of 1:1 acetonitrile/water containing 0.1% TFA. The protein isolation from the reaction mixture was repeated a few times to get enough material for analysis. The protein solutions eluted from the cartridge with 1:1 acetonitrile/water and 0.1% TFA were combined, evaporated to dryness by blowing with nitrogen at 50 °C, and stored in a freezer until analyzed. Gel Electrophoresis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) of proteins isolated from CFG was carried out on a Pharmacia Phast System (Piscataway, NJ) with a phast gel of 20% acrylamide. Dried protein samples were solubilized in 200 μL of protein solvent system (0.44 M Tris, 1 mM EDTA, and 10% SDS, pH 8.0) plus 40 μL of 2-mercaptoethanol (2-ME), and the mixtures were heated at 100 °C for 10 min. Gels were stained with 0.2% (w/v) Coomassie R350 dye. Molecular weight standards (Invitrogen Corp., Carlsbad, CA) and their corresponding molecular weights were as follows: phosphorylase, 98 kDa; bovine serum albumin (BSA), 62 kDa; glutamic dehydrogenase, 49 kDa; alcohol dehydrogenase, 38 kDa; carbonic anhydrase, 28 kDa; myoglobin red, 17 kDa; lysozyme, 14 kDa; aprotinin, 6 kDa; and insulin, B chain 3 kDa. Chymotrypsin Digestion of Protein Band from Gel. The protein bands from the gel electrophoresis were excised, destained, reduced with DTT, and alkylated with 2-iodoacetamide. Digestion of the protein bands was accomplished with chymotrypsin (Promega, Madison, WI), as a proteolysis enzyme, following the protocol from its manufacturer. The chymotryptic peptides were concentrated and cleaned using C18 resin pipet tips (ZipTip, Millipore Corp, Billerica, MA) by extracting them from the Zip Tip with a 5 μL solution of α-cyano-4hydroxy-cinnamic acid, 5 mg/mL in 1:1 acetonitrile/water containing
Figure 1. Corn fiber gum isolation scheme. 0.1% TFA (matrix solution), and spotted (1 μL) onto a stainless steel plate for mass spectrometry analysis. Mass Spectrometry and Protein Identification. The peptidespotted plate was analyzed with 4700 Proteomics Analyzer (Applied Biosystems, Framingham, MA), matrix-assisted laser desorption/ionization with automated tandem time-of-flight measurement of selected ions mass spectrometer (MALDI-TOF/TOF). The instrument was operated in the positive reflectron mode, averaging 1,000 acquired spectra in the mass range of 800 to 4,000 Da in the MS mode and 2,000 in the MS/MS mode. Conversion of TOF to mass (Da) for the monoisotopic ions, [M + H]+, was based on calibration of the instrument with a peptide standard calibration kit (Applied Biosystems). The MS and MS/MS results were combined and queried against the primary sequences from the National Center for Biotechnology Information (NCBI) database of nonidentical proteins using GPS Explorer Software (Applied Biosystems) with Mascot (Matrix Science, Inc. Boston, MA) as a search engine. The search criteria included 50 ppm and 0.1 Da errors in the MS and MS/MS mode, respectively, allowing for one missed chymotrypsin cleavage, oxidation of methionine, iodoacetamide alkylation of cysteine, and glutamine N-terminal pyroglutamylation as variable modifications. Reported protein matches are scored using the Mascot algorithm, which requires a score threshold of 72 with the NCBI database used for 95% confidence (p < 0.05).
’ RESULTS AND DISCUSSION CFG Isolation. Many investigators have published information on the composition of CFG, but only our group has extensively studied the presence of proteinous material associated with this arabinoxylan polysaccharide.1,6,7,13 As far as we know, there has been no formal report on the identity and kind of protein linked to CFG. For such protein identification, CFG was isolated from “fine” corn fiber following the scheme given in Figure 1. Fine corn fiber originates from the protein rich endosperm portion of the corn kernel and contains a comparatively higher percentage of protein in comparison to that of CFG isolated from pericarporiginating coarse fiber.7 In this study, to prepare lipid- and starch-free CFG, corn fiber was extracted with hexane to remove oil and treated with thermostable α-amylase at 90 95 °C to hydrolyze the starch present in the corn fiber. The CFG extraction was accomplished in 0.088 M NaOH and 0.026 M of Ca(OH)2 in the presence of 0.63% aqueous H2O2 at boiling temperature for 30 min while maintaining the pH at about 11.5 by adding more NaOH. The extraction time was reduced to 1/2 h from 1 h as given in our previous publications1,6 with the hope of preserving protein and other functional components on CFG with the shorter incubation time. The protein content of CFG 13290
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Figure 2. SDS polyacrylamide gel electrophoresis of protein obtained after CFG-deglycosylation and commercially available zein. Lane 1, protein molecular weight standards; lanes 2 and 3, proteins obtained after CFG deglycosylation (reduced), light and heavy load, respectively; lane 4, zein (not reduced); lane 5, zein (reduced).
isolated in the present conditions is comparatively higher (8.5 ( 0.05) than the previously isolated CFG (4 5%) from the same fiber source.7 CFG Deglycosylation and Gel Electrophoresis. Corn fiber gum proteins (CFG-proteins) obtained by TFMS deglycosylation of CFG and purification using C18 resin cartridges was characterized by SDS PAGE. Figure 2 shows an electrophoretic profile of CFG-proteins and a commercially available corn zein sample. CFG-proteins show very clear α-zeins bands at 19 23 kDa (lanes 2 and 3) matching with the bands in the standard commercial zein (lane 4), which also agree with the previous report.17,18 These CFG-proteins also show very light bands (too light to be seen in the figure) at 17 18 and 9 10 kDa (lane 3, heavy load), which are δ-zein and β-zein, respectively, as more visibly seen in the standard zein (lane 5). The two higher molecular weight bands of unreduced standard zein (lane 4) may be its dimers and tetramers, which on reduction become faint, but the α-zeins at 19 23 kDa (lane 5) become darker. There are a few light bands in the CFG-proteins (lane 3) between 28 and 49 kDa, which might be attributed to zein dimers and some other unknown proteins. In addition to zein proteins bands, CFG-proteins also show a smear of protein bands below 17 kDa, indicating the presence of some unknown low-molecularweight polypeptides. Chymotrypsin Digestion of the Protein Band and Mass Spectrometry Analysis. The intense protein bands from lanes 3 (CFG protein), 4, and 5 (zein proteins standard) with a MW of approximately 19 kDa (Figure 2) were excised, cleaned, and digested with chymotrypsin enzyme. This protease was selected because trypsin enzyme, which selectively cleaves proteins after lysine (K) or arginine (R), did not produce adequate tryptic peptides in the mass range analyzed. Chymotrypsin cleaves proteins specifically at the C-terminal of phenylalanine (F), leucine (L), tryptophan (W), and tyrosine (Y) unless it is followed by proline (P) in the sequence, which provided a better cleavage alternative for the protein digestions in this study. For simplicity, we will use the one letter amino acid abbreviation as shown above following the standard one letter code.19 The peptides purified by C18 resin pipet tips after chymotrypsin digestion were subjected to MALDI-TOF/TOF mass spectrometric analysis to produce the mass spectrum (MS) and the tandem fragmentation spectra
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(MS/MS) from the most intense selected ions in the MS (data not shown). The MS provided the masses of the chymotryptic peptides, and the MS/MS spectra gave specific fragmentation information for the peptide sequence elucidation. Consequently, the MS and the MS/MS spectra were combined and submitted for protein search in the NCBI database, limited to viridiplantea (green plants), using Mascot as a search engine. The proteinscoring algorithms used by Mascot rates proteins based on the probability that the identified proteins are a random hit within the database, and they are accepted as a significant match if the score values are with a confidence level of g95%. Accordingly, the protein threshold score value was 72 as determined by Mascot for the NCBI database used in this study. The MS and MS/MS combined database search of the chymotryptic peptides of the most intense protein bands on lanes 4 and 5 (Figure 2) produced identical spectra, returning three proteins matches above the threshold value. Only the matching results for the protein band of lane 4 are summarized in Tables 2 and 3 since the protein band from lane 5 has the same proteins hits with similar scores. Table 1 shows the matched peptides corresponding to a corn hypothetical protein entry in the database with a protein score of 94 and coverage of 26% of the database sequence. Tables 2 and 3 correspond to entries matching corn z1A alpha zein proteins with NCBI accession numbers gi:157780930 and gi:157780920, scores of 89 and 88, and sequence coverage of 42% and 39%, respectively. The last column in Tables 1 3 shows some modification to the indicated amino acid and whether it was confirmed by MS/MS. The quality of the MS/MS spectrum depends on the ability of the peptide to fragment and its concentration. It is often observed that only a few peptides produce good matching spectra. A common modification in peptides that have a Q at the N-terminal is the partial lactamization to form pyro-glutamate after gel digestion and processing, which causes the peptide to show very frequently in both forms. Also for the second entry in Table 2, the P in the matched sequence had a post-translational modification to hydroxyproline, a common modification expected in zein proteins.20,21 All of the identified sequences have common peptides, but the unique ones are highlighted in each table. The corresponding full database sequences for the proteins identified are presented in Table 4. It should be noted from Table 4 that the predicted MW for both z1A alpha zein proteins (Tables 2 3) are consistent with the gel band position at approximately 19 kDa (see MW markers in Figure 1, lane 1) but that it is shorter than the hypothetical corn protein (Table 1) which has a predicted MW of approximately 25 kDa (Table 4). This suggests that the mature form of the alpha zein protein is shorter than the hypothetical corn protein due to some processing or change during its maturation process. The MALDI-TOF/TOF analysis of the CFG-protein band produced a spectrum with peptides masses almost identical to the peptides identified for the zein standard. Figure 3 shows the back-to-back images for the MS of the chymotryptic peptides obtained from protein bands on lanes 4 (zein standard) and 3 (CFG-protein). The labeled peaks in Figure 3A are the peptides, which matched to the proteins in Tables 1 4, except for the peak at m/z = 1523.86 that corresponded to a chymotrypsin autolysis peptide. It is quite evident that the peptides produced from the CFG-protein band (Figure 3B) are consistent (within the 50 ppm of instrumental error) with the chymotryptic peptide profile of corn zein proteins (Figure 3A). The Mascot database search for the CFG-protein spectrum identified the same peptides as 13291
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Table 1. Matched Peptides for Hypothetical Corn Proteina calculated massb
observed massb
sequence positionc
sequencee
930.54
930.50
82 89
QQLPLVHL
1001.54
1001.50
164 171
QQQQLLPF
observationd Q as pyro-glutamate
1066.57
1066.56
224 233
QQHIIGGALF
confirmed by MS/MS
1083.59
1083.59
224 233
QQHIIGGALF
Q as pyro-glutamate confirmed by MS/MS
1131.56
1131.56
111 119
SQQHQFLPF
1140.65
1140.63
90 99
LVAQNIRAQQL
1393.85
1393.87
57 69
RIQQAIATGILPL
1691.02
1691.05
57 72
RIQQAIATGILPLSPL
confirmed by MS/MS
a
NCBI accession number, gi:195605670; protein scores, 94; sequence coverage, 26%. b The calculated and observed masses are the protonated form, [M + H]+, of the peptides. c The sequence position of the starting and ending amino acids are based on the database sequences presented in Table 4. d Under observation, the modification of specific amino acid is indicated and whether the sequence was confirmed by MS/MS spectrometry. e A = alanine; C = cysteine; D = aspartic acid; E = glutamic acid; F = phenylalanine; G = glycine; H = histidine; I = isoleucine; K = lysine; L = leucine; M = methionine; N = asparagine; P = proline; Q = glutamine; R = arginine; S = serine; T = threonine; Y = tyrosine; V = valine; W = tryptophan.
Table 2. Matched Peptides for z1A Alpha Zein Proteina calculated massb
observed massb
sequence positionc
sequencee
930.54 967.48
930.50 967.48
65 72 125 132
1001.54
1001.50
114 121
LQQQQLPF
1041.58
1041.59
74 82
AQNIRAQQL SQQQQFLPF
QQLPLVHL AVVYPQQF
1122.56
1122.56
94 102
1131.58
1131.56
156 166
NQLANVSPAAF
1154.66
1154.68
73 82
LAQNIRAQQL
1363.84
1363.86
40 52
RIQQAIAAGILPL
1410.78 1661.01
1410.81 1661.04
74 85 40 55
AQNIRAQQLQQL RIQQAIAAGILPLSPL
observationd Q as pyro-glutamate P as hydroxyproline
Confirmed by MS/MS
a
NCBI accession number, gi:157780930; protein score, 89; sequence coverage, 42%. b The calculated and observed masses are the protonated form, [M + H]+, of the peptides. c The sequence position of the starting and ending amino acids are based on the database sequences presented in Table 4. d Under observation, the modification of specific amino acid is indicated and whether the sequence was confirmed by MS/MS spectrometry. e A = alanine; C = cysteine; D = aspartic acid; E = glutamic acid; F = phenylalanine; G = glycine; H = histidine; I = isoleucine; K = lysine; L = leucine; M = methionine; N = asparagine; P = proline; Q = glutamine; R = arginine; S = serine; T = threonine; Y = tyrosine; V = valine; W = tryptophan.
Table 3. Matched Peptides for z1A Alpha Zein Proteina calculated massb
observed massb
sequence positionc
sequencee
827.50
827.48
153 159
LPQLLPF
930.54
930.50
65 72
QQLPLVHL
1001.54
1001.53
114 121
LQQQQLPF
1041.58
1041.59
74 82
AQNIRAQQL
1122.56
1122.56
94 102
SQQQQFLPF
1154.66
1154.68
73 82
LAQNIRAQQL
1363.84 1410.78
1363.86 1410.81
40 52 74 85
RIQQAIAAGILPL AQNIRAQQLQQL
1606.83
1606.86
122 135
SQLPAAYPQQFLPF
1661.01
1661.04
40 55
RIQQAIAAGILPLSPL
observationd
Q as pyro-glutamate
confirmed by MS/MS
a
NCBI accession number, gi:157780920; protein score, 88; sequence coverage, 39%. b The calculated and observed masses are the protonated form, [M + H]+, of the peptides. c The sequence position of the starting and ending amino acids are based on the database sequences presented in Table 4. d Under observation, the modification of specific amino acid is indicated and whether the sequence was confirmed by MS/MS spectrometry. e A = alanine; C = cysteine; D = aspartic acid; E = glutamic acid; F = phenylalanine; G = glycine; H = histidine; I = isoleucine; K = lysine; L = leucine; M = methionine; N = asparagine; P = proline; Q = glutamine; R = arginine; S = serine; T = threonine; Y = tyrosine; V = valine; W = tryptophan.
present in the zein standard bands, but the scores were in the order of 62 (∼95% confidence). The database search was affected by the low quality of the MS/MS spectrum of the CFG
peptides due to a limited sample available as reflected in the low concentration of the protein in the corresponding gel band. However, this finding demonstrates very clearly that the chymotrypsin 13292
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Table 4. NCBI Matched Proteins for the More Intense Gel Band in Lane 4, Figure 2 Hypothetical Corn Protein (NCBI Accession Number: gi:195605670, MW = 25,439 sequencea
position 1 50
MAAKIFCFLM
LLGLSASVAT
ATIFPQCSQA
PIASLLPPYL
SPAVSSMCEN
51 100
PIVQPYRIQQ
AIATGILPLS
PLFLQQPSAL
LQQLPLVHLV
AQNIRAQQLQ
101 150
QLVLANLAAY
SQQHQFLPFN
QLAALNSAAY
LQQQLPFSQL
VAAYPQQFLP
151 200
FNQLAALNSA
AYLQQQQLLP
FSQLADVSPA
AFLTQQQLLP
FYLHAMPNAG
201 233
TLLQLQQLLP
FNQLALTNST
ASYQQHIIGG
ALF
Z1A Alpha Zein Protein (NCBI Accession Number: gi:157780930 MW = 18,594 sequence
position 1 50
AATATIFPQC
SQAPIASLLP
PYLSPAVSSV
CENPILQPYR
IQQAIAAGIL
51 100
PLSPLFLQQS
SALLQQLPLV
HLLAQNIRAQ
QLQQLVLANL
AAYSQQQQFL
101 150 151 170
PFNQLGSLNS QLPPFNQLAN
ASYLQQQQLP VSPAAFLTQQ
FSQLAVVYPQ
QFLPFNQLAT
LNSAAYLQQQ
Z1A Alpha Zein Protein (NCBI Accession Number: gi:157780920 MW = 18,696 sequence
position 1 50
AATATIFPQC
SQAPIASLLP
PYLSPVVSSV
CQNPILEPYR
IQQAIAAGIL
51 100
PLSPLFLQQS
SALLQQLPLV
HLLAQNIRAQ
QLQQLVLANL
AAYSQQQQFL
101 150 151 171
PFNQLAALNS QLLPQLLPFY
ASYLQQQQLP QHAAPNAGTL
FSQLPAAYPQ L
QFLPFNQLAA
LNSPAYLQQQ
a
A = alanine; C = cysteine; D = aspartic acid; E = glutamic acid; F = phenylalanine; G = glycine; H = histidine; I = isoleucine; K = lysine; L = leucine; M = methionine; N = asparagine; P = proline; Q = glutamine; R = arginine; S = serine; T = threonine; Y = tyrosine; V = valine; W = tryptophan.
Figure 3. MALDI-TOF/TOF mass spectra of the chymotrypsin peptides from the electrophoresis gel in Figure 2. A: peptides corresponding to the intense gel band in lane 4. B: peptides corresponding to the CFG-protein gel band in lane 3.
peptides obtained from CFG are highly consistent with those in commercial corn zein. According to the gel position in Figure 2 and spectra in Figure 3, it seems reasonable to conclude that proteins found in CFG are mainly the same protein identified in the commercial corn zein protein with a MW of approximately 19 kDa. Scientists have known that CFG is a good emulsifier for over 50 years, but none has been able to explain how a hydrophilic
arabinoxylan polysaccharide would be able to function as a good oil-in-water emulsifier without the presence of hydrophobic groups that could interact with oil droplets. The finding here that protein strongly associated with CFG, either covalently or physically, may be similar to the hydrophobic protein zein could finally explain CFG’s excellent emulsifier functionality. 13293
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’ AUTHOR INFORMATION Corresponding Author
*Tel: 215-836-3783. Fax: 215-233-6406. E-mail: madhav.yadav@ ars.usda.gov.
’ ACKNOWLEDGMENT We thank Stefanie Simon, John Minutolo, and Laurie Fortis for their technical support and ADM Research for providing the sample of corn “fine fiber”.
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(19) IUPAC-IUB Commission on Biochemical Nomenclature (CBN). A one-letter notation for amino acid sequences. Arch. Biochem. Biophys. 1988, 3, 125, i v. (b) Moss, G. P. IUPAC-IUB: Nomenclature and Symbolism for Amino Acids and Peptides, 1983, world wide web version. http:/www.chem.qmul.ac.uk/iupac/AminoAcid/. (20) Hood, E. E.; Shen, Q. X.; Varner, J. E. A developmentally regulated hydroxyproline-rich glycoprotein in Maize pericarp cell walls. Plant. Physiol. 1998, 87, 138–142. (21) Anderson, D. M. W.; Howlett, J. F.; McNab, C. G. A. The amino acid composition of the proteinaceous component of gum arabic (Acacia senegal (L.) wild. Food Addit Contam. 1985, 2, 159–164.
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