不同花生品种籽仁发育过程中蛋白质组分分析

采用蛋白质组分的连续累进提取法提取4个花生品种籽仁的清蛋白、球蛋白、醇溶蛋白和谷蛋白。利用聚丙烯酰胺凝胶电泳(SDS-PAGE)技术,对各蛋白组分的亚基组成进行分析。结果表明,花生籽仁发育过程中,清蛋白始终占绝对优势,占花生蛋白质总量的70%以上,最高达95%;球蛋白含量较低,仅占花生蛋白质总量的3%~5%,谷蛋白含量最低,其不足3%,醇溶蛋白痕量;在4种组分中,品种之间球蛋白含量差异最大。SDS-PAGE图谱显示,染色后清蛋白、球蛋白条带清晰可见,谷蛋白条带较为模糊,清蛋白含量高,清蛋白和球蛋白成分较丰富。花生蛋白亚基分子质量在14.4~97.4 ku之间,清蛋白有10~12个亚基,球蛋白有9~10个亚基。采用连续累进提取法可将花生蛋白组分分离,利用SDS-PAGE方法可以得到花生籽仁清蛋白、球蛋白的清晰条带且多态性高,而谷蛋白条带模糊。     

未成熟蚕豆蛋白组分的分析

通过分析5个未成熟蚕豆品种在3个可食用阶段的蛋白质组分,探索未成熟蚕豆种子蛋白质组分的变化规律。采用分级提取的方法分别提取清蛋白、球蛋白、醇溶蛋白和谷蛋白组分,并利用考马斯亮蓝法和SDS-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)对其含量和组分进行测定。结果表明,5个蚕豆品种最主要的蛋白组分为谷蛋白,其次为清蛋白和球蛋白,醇溶蛋白含量最低;随着蚕豆种子的不断成熟,清蛋白和球蛋白的含量明显增加;醇溶蛋白含量明显减少;谷蛋白含量因品种的不同,而表现出多样性;同一可食阶段,不同品种蛋白组分含量具有差异;随着可食阶段的延后,清蛋白和球蛋白的SDS-PAGE电泳条带逐渐增多。随着蚕豆种子的不断成熟,其蛋白各组分的含量发生变化:从S1到S2阶段,清蛋白的40 kDa的条带变浅,新增15 kDa至35 kDa的条带,球蛋白中的40 kDa的条带变浅甚至消失,新增20 kDa至35 kDa以及55 kDa的条带;从S2到S3阶段,清蛋白的15 k Da至35 k Da的小分子量条带颜色变浅,其含量减少,出现了60 kDa～65 kDa和70 kDa～85 kDa,甚至更大分子量的条带,而球蛋白的15 kDa至35 k Da的小分子量条带颜色变浅,40 kDa条带再次出现且加深,出现了60 kDa～65 kDa和70 kDa～85 k Da,甚至更大分子量的条带。这些表明在蚕豆成熟过程中,一些蛋白到一定阶段被降解,一些新的蛋白又被合成。     

花椒籽壳蛋白与籽仁蛋白理化性质分析

以花椒籽为原料,主要研究花椒籽仁与籽壳脱脂后的基本化学成分、蛋白等电点、氨基酸组成、亚基组成以及蛋白组分等理化性质的差异。结果显示,脱脂后的花椒籽仁和籽壳中蛋白质含量分别为61. 17%、8. 54%,其蛋白等电点分别为3. 5、4. 5。花椒籽仁脱脂粉的氨基酸总量为43g/100 g,必需氨基酸占总量的27. 31%,籽壳脱脂粉的氨基酸总量为4. 07 g/100 g,必需氨基酸占总量的26. 78%,两者主要的氨基酸为天冬氨酸、谷氨酸和精氨酸。采用Osborne法对蛋白分类,花椒籽仁中清蛋白、球蛋白、谷蛋白、醇溶蛋白的含量分别为7. 76%、72. 06%、15. 80%、1. 22%,籽壳中相对应的蛋白含量分别为13. 12%、56. 54%、21. 45%、3. 25%。SDS-PAGE电泳分析表明:花椒籽仁粗蛋白有7个条带,相对分子质量在0～66. 200 k Da之间,籽壳粗蛋白有2个条带,相对分子质量在18. 400～35. 000 k Da之间;花椒籽仁4种蛋白组分相对分子质量较小,亚基相对分子质量分布在0～45 k Da之间,花椒籽壳4种蛋白组分分布范围较小,亚基相对分子质量分布在14. 4～35 k Da之间。     

4种花生粕蛋白的理化性质及功能特性研究

以花生粕为原料,采用分级提取工艺提取花生清蛋白、球蛋白、醇溶蛋白和谷蛋白,研究4种花生粕蛋白的理化性质和功能特性。扫描电镜观察,4种花生粕蛋白的形态结构各不相同。SDS-PAGE法测定分子质量表明,清蛋白含有4种亚基,分子质量为70、40、30、25和15 ku;醇溶蛋白含有2种亚基,分子质量分别为25和1 5 ku;球蛋白含有5种亚基,相对分子质量分别为40、38、30、25和15 ku;谷蛋白含有4种亚基,相对分子质量分别为40、30、25和15 ku。花生清蛋白、醇溶蛋白、球蛋白、谷蛋白的等电点分别为pH 3.6、pH 5.2、pH 4.6、pH 5.0。功能性质研究表明,球蛋白的持水性最好,为1.52 mL/g,其次为谷蛋白1.10 mL/g,清蛋白和醇溶蛋白的持水性较低分别为0.49、0.14 mL/g;清蛋白的持油量相对较高为8.21mL/g,其次为球蛋白为7.16 mL/g,谷蛋白和醇溶蛋白的持油量相对较低,分别为3.82 mL/g和5.49 mL/g;清蛋白的乳化性和乳化稳定性相对较高,乳化能力(EC)值和乳化稳定性(ES)值分别为7 1.4%和83.33%,谷蛋白次之,EC和ES值分别为66.7%和82.86%,醇溶蛋白和球蛋白相对较低,EC值分别为64.0%和62.2%,ES值分别为82.35%和76.67%。综上,花生粕清蛋白的持油性、乳化性和乳化稳定性相对较好。     

即食小米粥贮藏过程中蛋白组分变化规律的研究

采用Osborne法对小米及即食小米粥中的清蛋白、球蛋白、醇溶蛋白、谷蛋白进行提取,运用聚丙烯酰胺凝胶电泳法对小米以及贮藏不同时间的即食小米粥中的4种蛋白质进行亚基分析。通过对比小米和即食小米粥蛋白组分发现,即食小米粥中4种蛋白组分的含量都有不同程度的减少,其中醇溶蛋白降幅最大,其次是清蛋白、球蛋白;由电泳图谱可知,即食小米粥加工工序对小米中清蛋白、球蛋白的影响最大,清蛋白和球蛋白的部分亚基随废水排出而损失,剩余亚基通过二硫键形成新的蛋白组分,并以高分子聚集体的形式保存在即食小米粥中。随贮藏时间的延长,即食小米粥中清蛋白、球蛋白、醇溶蛋白和谷蛋白含量及亚基数量均有不同程度的减少,到贮藏12 d时,清蛋白、球蛋白和谷蛋白的亚基条带几乎全部消失。     

脱脂小麦胚芽蛋白分类及其氨基酸组成分析

对脱脂小麦胚芽蛋白进行Osboren分类研究,进一步研究了各蛋白组分的亚基分子质量分布、氨基酸组成和营养价值.试验结果表明,球蛋白和清蛋白是小麦胚芽蛋白的主要组分.SDS-PACE分析中,清蛋白主要亚基分子质量为15 000、17 400、20 500、29 000、33 400～37 100和54 900 u.球蛋白主要亚基分子质量为12 300、20 000、23 500、36 000～40 100和47 000～55 200 u,谷蛋白的主要亚基分子质量为55 000、41 800和＜27 000 u.同时,试验结果也表明,清蛋白的氨基酸评分最高,其次分别为谷蛋白、球蛋白和醇溶蛋白;清蛋白具有最高的蛋白质效率比(PER)和体外消化率(IVPD),醇溶蛋白和谷蛋白的PER和IVPD较小;清蛋白的蛋白质消化率校正的氨基酸记分法(PDCAAS)最高,球蛋白、醇溶蛋白和谷蛋白的PDCAAS较小.因此,与FAO/WHO模式相比,清蛋白具有较好的氨基酸组成和PDCAAS,是一种优质蛋白质.     

大红袍花椒籽种仁蛋白的分类研究

对大红袍花椒种仁蛋白质进行了Osborne蛋白质分类,分离出花椒种仁清蛋白16.38%、球蛋白41.09%、醇溶蛋白2.53%、谷蛋白33.29%,然后将分类后的各种花椒种仁蛋白质进行SDS-PAGE电泳分析测定亚基大小.结果表明,蛋白质组分中含有大量盐溶性的球蛋白和碱溶性的谷蛋白,易采用盐溶法或稀碱提取;并且花椒种仁中各蛋白质组分的相对分子质量较小,有利于人体对蛋白质的消化吸收.     

谷子蛋白组分分析研究

以谷子为研究对象,采用Osbron方法提取谷子蛋白组分,确定不同蛋白组分等电点,分析不同品种谷子蛋白组分差别。结果显示:谷子清蛋白、球蛋白、谷蛋白、醇蛋白等电点分别为3.8,3.6,4.4,5.6;14个品种谷子清蛋白、球蛋白、谷蛋白、醇蛋白、其他蛋白和总蛋白的平均值分别为0.34,1.17,1.39,1.22,7.42,11.54 g/100 g;蛋白组分分布差异由大到小依次为:清蛋白、醇蛋白、球蛋白、谷蛋白、其他蛋白和总蛋白;通过系统聚类方法可将14个品种谷子分成3类,分别含有6、5和3个品种,聚类结果显示品种所处产地对蛋白质组分有重要影响。     

燕麦蛋白组分分离提取及其SDS-PAGE电泳分析

通过单因素实验对燕麦蛋白组分的分离提取工艺进行了优化,并通过SDS-PAGE电泳对燕麦蛋白组分进行亚基分析。结果表明:燕麦清蛋白提取的最佳温度为40℃,球蛋白提取最佳盐浓度为7%,醇溶蛋白提取的最佳乙醇浓度为75%,谷蛋白提取的最佳碱浓度为0.05 mol/L,蛋白质提取率为83.1%。SDS-PAGE实验结果显示:燕麦清蛋白在10~100 kD范围内均有分布,燕麦球蛋白由2个亚基组成,分子量分别在97.4~100 kD和43~66.2 kD范围内,燕麦醇溶蛋白亚基大部分集中在18.39~40.72kD之间,燕麦谷蛋白部分亚基分布在20.67~26.66kD与43.29~50.80 kD之间。     

4种花生粕蛋白的理化性质及功能特性研究

以花生粕为原料,采用分级提取工艺提取花生清蛋白、球蛋白、醇溶蛋白和谷蛋白,研究4种花生粕蛋白的理化性质和功能特性。扫描电镜观察,4种花生粕蛋白的形态结构各不相同。SDS-PAGE法测定分子量表明,清蛋白含有4种亚基,相对分子量分别为70kDa、40kDa、30kDa、25kDa和15kDa;醇溶蛋白含有2种亚基,分子量分别为25kDa和15kDa;球蛋白含有5种亚基,分子量分别为40kDa、38kDa、30kDa、25kDa和15kDa;谷蛋白含有4种亚基,分子量分别为40kDa、30kDa、25kDa和15kDa。花生清蛋白、醇溶蛋白、球蛋白、谷蛋白的等电点分别为pH3.6、pH5.2、pH4.6、pH5.0。功能性质研究表明,球蛋白的持水性最好,为1.52mL/g,其次为谷蛋白1.10mL/g,清蛋白和醇溶蛋白的持水性较低分别为0.49mL/g、0.14mL/g;清蛋白的持油量相对较高为8.21mL/g,其次为球蛋白为7.16mL/g,谷蛋白和醇溶蛋白的持油量相对较低,分别为3.82mL/g和5.49mL/g;清蛋白的乳化性和乳化稳定性相对较高,乳化能力EC值和乳化稳定性ES值分别为71.4% 和83.33%,谷蛋白次之,EC和ES值分别为66.7% 和82.86%,醇溶蛋白和球蛋白相对较低,EC值分别为64.0% 和62.2%,ES值分别为82.35% 和76.67%。综上,花生粕清蛋白的持油性、乳化性和乳化稳定性相对较好。     

Characterization of protein fractions from chickpea (Cicer arietinum L.) and oat (Avena sativa L.) seeds using proteomic techniques

Albumin, globulin and glutelin fractions were prepared from chickpea and oat seeds using sequential extractions. Molecular characteristics of individual protein fractions were investigated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in combination with proteomic techniques. SDS-PAGE results revealed that chickpea albumin and globulin fractions (C-Ab and C-Gb) showed protein bands with molecular weights (MWs) related to subunits of legumin (11S globulin) and pea vicilin (7S globulin); oat protein fractions (O-Ab, O-Gb and O-Gt) showed most protein bands with MWs related to subunits of oat 12S globulin (avenalin). With proteomic analysis, eighteen tryptic peptides from chickpea globulin fraction showed sequence homology that corresponded to chickpea legumin α- and β-subunit (NCBI accession number: gi|6273402; theoretical mass 56,216 Da) while sixteen tryptic peptides from chickpea albumin fraction (C-Ab) were identified as chickpea provicilin precursor (NCBI accession number: gi|82173888; theoretical mass 51,390 Da); fifteen tryptic peptides from oat protein fractions were identified with origin from oat 12S seed storage globulin 1 (NCBI accession number: gi|134918; theoretical mass 58,508 Da). The identified tryptic peptide, ALIVPQNFAIAAK, was commonly found in chickpea glutelin fraction (C-Gt), rice glutelin fraction (R-Gt), and oat albumin, globulin and glutelin fractions (O-Ab, O-Gb and O-Gt).     

苦荞蛋白质的低消化性研究Ⅱ——酶解产物的超微结构分析和分子量分布

苦荞麦蛋白质氨基酸组成平衡,生物价高,并且具有独特的生理功能。体外消化实验表明其蛋白质的消化率较低,通过扫描电镜对四种蛋白质组分酶解产物的超微结构进行观察发现,胃蛋白酶作用于四种组分的方式是不同的,胃蛋白酶不仅可以作用与清蛋白和球蛋白的表面,而且随着水解进程的延长胃蛋白酶还可以作用与清蛋白和球蛋白的内部结构,因此其体外消化率相对较高。而对于醇溶蛋白和谷蛋白,其高级结构相对较为稳定,胃蛋白酶只能作用于其表面,很难作用其内部结构,所以这两种蛋白组分的体外消化率相对较低。此外还通过高效液相对其酶解物的分子量分布进行研究,结果表明,清蛋白和球蛋白的酶解产物分子量相对较低,组分多,酶解程度高。醇溶蛋白酶解物的组成较为简单,这可能是由于被胃蛋白酶作用的位点较少所造成的。而谷蛋白,其酶解物的分子量分布广,高分子量的组分所占比例大,这表明其被酶解的程度相对较低。     

ISOLATION, PURIFICATION AND CHARACTERIZATION OF THE SEED STORAGE GLOBULIN AND ITS POLYMERIZED FORM FROM TRITICUM AESTIVUM

The salt-soluble globulin and its polymerized counterpart from wheat (Triticum aestivum) seed were isolated and purified to homogeneity employing both gel filtration and anion exchange chromatography. The two globulins were purified free of any purothionin (an oxidation-reduction protein) contamination. Some of the physico-chemical properties of purothionin are also reported. Both globulins were found to be oligomers composed of two polypeptide chains, i.e., 35,000 and 49,000 Da, with no apparent evidence of any covalent inter-chain disulfide linkages between these major subunits. The molecular weights for the globulin and its polymerized (i.e., polymerized via interchain disulfide bonds between various subunits of differing molecular weights, excluding any interchain disulfide bonds between the 35,000 and 49,000 Da subunits) counterpart were determined to be 474,000 and 567,000 Da, respectively, by gel chromatography. Surface charge profiles of fractions 2 and 3 were determined using electrophoretic isoelectric focusing, electrophoretic titration and zeta potential analysis and indicated pIs of 6.90 and 6.55, respectively. The nonpolymerized globulin was found to be more positively charged below its isoelectric point and less negatively charged above it than its polymerized counterpart. Microcalorimetric studies indicated that the two globulins had similar T_m values.     

Isolation and Characterization of Chickpea (Cicer arietinum L.) Seed Protein Fractions

Proteins of ground chickpea seeds were extracted with sodium hydroxide (NaOH) solution and precipitated with addition of acid (isoelectric precipitate (C-IP)) and by cryoprecipitation (cryoprecipitate (C-CP)). The protein isolates were characterized by Native PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), reversed-phase high performance liquid chromatography (RP-HPLC) and electrospray-ionization mass spectrometry (ESI/MS). Both the isoelectric precipitate and cryoprecipitate contained the globulin protein 11S legumins and 7S vicilins as the major protein fractions and 2S albumin proteins as a minor protein fraction. The major subunits of RP-HPLC protein fractions from both cryoprecipitate and isoelectric precipitate were found to contain subunits of both legumins and vicilins. SDS-PAGE identified legumin α-subunits with MW 40.6 and 39.5 kDa and legumin β- subunits with MW 23.5 and 22.5 kDa, and vicilin subunits with MW 70.2, 50.7, 35.0, 33.6, 18.9 and 15.5 kDa. ESI-MS molecular weights 35,366, 35,268 and 14,648 Da are likely vicilin subunits while the 24,894 Da is a legumin β-subunit.     

Changes in the protein complex of wheat dough affected by soybean 11 S globulin: Part 2—The interactions of soybean 11 S globulin with gluten proteins

The interactions between soy 11 S globulin and protein fractions of wheaten doughs were examined using a multistep extraction method followed by molecular sieving and characterisation of the fractions obtained. The soy 11 S globulin preparation was coupled with fluoresceine isothiocyanate to permit tracing of this protein in the complexes formed. In model experiments the average molecular weights of globulin/prolamine complexes were determined, the dispersion of light on the protein molecules being measured. The formation of high molecular weight complexes of soy 11 S globulin and prolamine was found. A shift between albumin/globulin and gluten fractions was also observed, which resulted in an increase in the contents of low molecular weight fractions dispersible in pyrophosphate buffer and acetic acid.Most of the investigations concerning the enrichment of bread with soybean proteins deal with technological problems. The documentation of the chemically induced changes in the dough-protein complex is rather limited and a number of questions are still awaiting answers. The data reported by Jakubczyk et al. (1973), Matthews (1972), Pollock & Geeds (1960a, b) do not explain the character of the interactions between soy proteins and gluten systems and give only inadequate information on the changes of molecular structure or aggregation/disaggregation phenomena occurring in the proteins of enriched dough. The results presented in our previous study (Lampart-Szczapa & Jankiewicz, 1982, encouraged us to continue the experiments to explain the rôle played by soybean 11 S globulin treated as a model soy protein in modifying the gluten matrix of wheat dough. In this paper the effects of soybean 11 S globulin on the fractional distribution of the dough-protein complex and average molecular weights of the fractions are presented.     

Osborne法分级提取五味子蛋白及抗氧化活性比较

目的:采用Osborne法提取五味子各组分蛋白,并对其体外抗氧化活性比较研究。方法:用Osborne方法分级提取五味子中的蛋白质,得到四种组分蛋白:清蛋白、球蛋白、醇溶蛋白和谷蛋白,对其进行十二烷基硫酸钠-聚丙烯酰胺凝胶（sodium lauryl sulfate-polyacrylamide gel,SDS-PAGE）电泳分析,并对不同组分蛋白和总蛋白的体外抗氧化活性进行对比研究。结果:分级提取得到的四种蛋白质,清蛋白亚基分子量分布在15~25 kDa和40~55 kDa,球蛋白分子量大致分布在15~25 kDa和35~55 kDa,谷蛋白亚基分子量在15~20 kDa和30~40 kDa。以五味子总蛋白为参照物,体外抗氧化试验结果表明,总蛋白对羟基自由基和DPPH自由基的清除能力强于各分级蛋白;四种分级蛋白对Fe3+的还原能力强于总蛋白;在ABTS自由基清除试验中含量最高的谷蛋白效果最佳,其清除效果在一定的浓度下与Vc接近,同时,其在羟基、DPPH自由基与Fe3+还原能力的试验中抗氧化活性良好,表明五味子组分蛋白中谷蛋白可作为一种良好的抗氧化潜在物质。     

Molecular weight and amino acid composition of glycinin subunits

Glycinin, the major soyabean globulin, is composed of subunits having molecular weights of about 22,300, and 37,200. On the basis of amino acid analysis, the six submits of glycinin isolated by isoelectric focusing are all different. The ‘acidic’ subunits have higher content of glutamic acid and proline, whereas the ‘basic’ subunits are higher in the hydrophobic amino acids leucine, tyrosine, phenylalanine, valine and alanine.     

Chemical investigation of Erythrina variegata Linn. seed proteins

Proteins were extracted from deoiled seeds of Erythrina variegata Linn., a potential source of non-conventional seed, in aqueous solutions of various pHs or by different concentrations of NaCl, KCl, CaCl₂ and MgSO₄ at pH 7.0. Nitrogen contents of the seeds and deoiled seeds showed good protein content. Fractionation of protein was done to separate albumin, globulin, prolamine and glutelin. Amino acid analysis of the total protein isolates (TPI) and the fractions isolated (except prolamine) identified 17 amino acids, most of which were essential. The molecular weights of TPI and the fractions were determined by SDS–PAGE electrophoresis. The results showed that TPI was composed of twelve bands, eight for globulin, nine for prolamine and six for glutelin. Studies on surface structure of the proteins and seed flour by scanning electron microscopy (SEM) are also included.     

Purification and characterisation of seed globulins from black gram (Phaseolus mungo)

The salt-soluble proteins and crude globulins of black gram (Phaseolus mungo, Rox b), using crossed immunoelectrophoresis for identification, were separated into 28–29 and seven to eight individual components, respectively. Three major globulins (G1, G2 and G3), of which G1 was identified as a glycoprotein, were purified by anionexchange chromatography followed by gel filtration. Fused rocket immunoelectrophoresis was used for the localisation of these proteins in the different fractions. In crossed immunoelectrophoresis, purified G1 and G2 globulins each showed a single peak, while G3 globulin still contained four to five distinct peaks. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed three subunits for G1 globulin (molecular weight 64 500, 55 000 and 50 000), three distinct subunits for G2 globulin (mol. wt. 67 000, 60 000 and 16 000), and six distinct subunits for G3 globulin (mol. wt 50 000, 42 000, 28 000, 26 000, 21 000 and 10 000). Isoelectric focusing in 6m urea showed three acidic subunits for G1 globulin. G2 subunits were separated into three acidic and two basic subunits. G3 globulin contained about ten acidic subunits. Immunochemical examination of pure G1 globulin indicated microheterogeneity similar to that found for vicilin extracted from other legume seeds. Based on molecular weights, subunit structure and amino acid composition, the G1 globulin of black gram was found to correspond with vicilin (glycoprotein II), and G2 globulin to legumin of other legume seed proteins. G1 and G3 globulins were found to be major storage proteins in black gram.     

Extraction and characterization of chickpea (Cicer arietinum) albumin and globulin

ABSTRACT:  Albumin and globulin fractions of 1 Desi and 2 Kabuli varieties of chickpeas ( Cicer arietinum ) were extracted with water and salt solutions (K₂SO₄ and NaCl). The extractable yields and particularly the albumin-globulin ratio varied greatly with the extraction medium and chickpea variety. Depending on the procedure employed, albumin could be extracted as a major fraction of chickpea proteins. Higher levels of essential amino acids and sulfur containing amino acids were found in albumins than in globulins of all chickpeas investigated. The common structural characteristics of both Kabuli and Desi chickpea albumins and globulins were clearly identified by densitometric profiles of their sodium dodecyl sulfate polyacrylamide gel patterns. Albumins contained subunits with higher molecular weights than those of globulins. The in vitro digestibility of the chickpea proteins by papain, pepsin, chymotrypsin, and trypsin indicated that globulins were more susceptible to proteolytic hydrolysis.