m</sub>) 測(cè)定是生物化學(xué)實(shí)驗(yàn)教學(xué)大綱中的綜合性實(shí)驗(yàn)課程內(nèi)容。本次實(shí)驗(yàn)實(shí)踐以市售綠豆芽莖段為材料，利用研磨法提取其總蛋白，采用BCA法、SDS-PAGE電泳法測(cè)定總蛋白濃度和分布；以對(duì)硝基苯磷酸二鈉( PNPP-Na<sub>2</sub>) 為底物，在酸性條件[37 °C,0.2mol/L HAc-NaAc緩沖液 (pH5.6) ]下測(cè)定了總蛋白中酸性磷酸酶的米氏常數(shù)。結(jié)果表明，綠豆芽莖段總蛋白濃度為 4.374mg/mL ，在酸性條件下，總蛋白中酸性磷酸酶的 K<sub>m</sub> 為 5.5525mmol/L ，酶促反應(yīng)最大速度( ∣V<sub>m</sub>) 為 0.0743mmol/(L?min) 。該實(shí)踐強(qiáng)化了學(xué)生對(duì)蛋白質(zhì)提取分離、定量分析及米氏常數(shù)測(cè)定等基礎(chǔ)理論知識(shí)的理解，使其掌握了蛋白質(zhì)提取分離和SDS-PAGE電泳等實(shí)驗(yàn)技術(shù)，并激發(fā)了其對(duì)科學(xué)研究的興趣和熱情。-龍?jiān)雌诳W(wǎng)" />

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蛋白質(zhì)提取分離分析及米氏常數(shù)測(cè)定的實(shí)踐及效果

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中圖分類號(hào) G642.1 文獻(xiàn)標(biāo)識(shí)碼A 文章編號(hào) 1007-7731（2025）13-0118-04

DOI號(hào)10.16377/j.cnki.issn1007-7731.2025.13.030

Practice and effect of protein extraction， separation， analysis， and Michaelis constant determination

JIA Haoran HAN Peng ZHANG Yantao ZHANG Liquan （CollegeofLife Sciences，Inner Mongolia University，HohhotO1oO21， China）

AbstractProtein extraction，separation，analysis and determination of Michaelis constant are the comprehensive experimental coursecontents inthe teaching sylabusofbiochemistryexperiments.In thisexperimental practice， commercially available mung bean sprout stem segments were used as experimental materials.The total protein was extracted bygrinding method，andtheconcentrationand distributionof total protein were determinedby BCA method and SDS-PAGE electrophoresis.The Michaelis constant of acid phosphatase in total protein was determined using disodium p-nitrophenyl phosphate （PNPP-N ）as the substrate under acidic conditions[37 °C ，0.2 mol/L HAc-NaAc α bufr （pH=5.6）.The results showed that the total protein concentration of mung bean sprout stem segments was 4.374 mg/mL.Under acidic conditions，the Km of acid phosphatase in the total protein was 5.552 5 mmol/L，and the Vm was 0.0743mmol/（L·min）.This practical exercise significantly enhancedstudents’operational understandingof fundamentalbiochemicaltechniques including protein extraction/purification，quantitative analysis，and determination of Michaelis constant.Furthermore，it provided hands-on experience with esential methodologies such asprotein isolationand SDS-PAGE electrophoresis， while efectively fostering scientific curiosityand research enthusiasm among participants.

Keywords Biochemistry; extraction and determination of protein; Michaelis constant; mung bean sprouts

蛋白質(zhì)是高校生物化學(xué)課程的核心教學(xué)內(nèi)容之一。（剩余5522字）

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