| Abstract: | 在不同濃度(0.0M , 0.01M , 0.025M , 0.1M , 0.5M)的磷酸鹽緩衝液（起始pH值為7.5）下以100℃水浴加熱1M的glucose與glycine溶液；結果磷酸鹽緩衝液濃度越高的組別反應後的pH值下降程度越高，形成的梅納反應產物(maillard reaction products, MRPs)濃度（由褐變指數Browning Index表示）也越高，而且其DNA斷裂活性也越強烈。相同的，1M的glucose與glycine溶液在不同濃度(0.0M, 0.01M, 0.025M, 0.5M, 0.1M, 0.5M, 1.0M)的磷酸鹽緩衝液（起始pH值為7.5）下儲存於25℃暗室中，隨著儲存時間增加，磷酸鹽緩衝液濃度越高的組別形成的MRPs濃度也越高，DNA* 以在相同莫耳濃度的3-(N-Morpholino)pro-panesulfonic acid(MOPS),N,N-Bis-(2-hydroxyethyl)2-aminoethane-sulfonic acid(BES),與磷酸鹽等緩衝液下（三者提供的pH值緩衝能力相仿，其pKa值皆在7.2左右）反應得到的MRPs作比較；磷酸鹽緩衝液確實會比MOPS、BES等緩衝液更能促進所形成的MRPs具有更高的DNA斷裂性質。控制一定濃度的磷酸鹽緩衝液在不同起始pH值(pH 5.5 , 6.5 , 7.5 , 8.5 , 9.5)下加熱反應得到MRPs，發現起始pH值越低越不利於MRPs的生成（起始pH值越低反應後的溶液之褐變指數越低），但是對DNA斷裂活性卻無顯著的影響。 以分子濾膜過濾分析在1M磷酸鹽緩衝液(起始pH 7.5)下加熱(at 100℃)1小時的1M Glucose-Glycine溶液，發現所得到的[陳景榮1]MRPs絕大部分的分子量都集中在MW 5,000以下的濾液內，此濾液再經Sephadex G-25 column膠體層析後，會清楚分成兩帶；且至少有一帶的次濾液(帶一)具有DNA斷裂活性。同時再以灰化等測磷的方法分析次濾液中的總磷含量；發現總磷值波峰出現的位置與帶一並不完全吻合；且後來經實驗證實，此時出現的磷屬於無機磷，亦即可能未與MRPs結合。 另外，在純水(起始pH 7.5)下加熱(at 100℃)24小時的1M Glucose-Glycine溶液(MRPs(H))，其褐變指數雖然近似於在1M磷酸鹽緩衝液下加熱1小時 (at 100℃、起始pH 7.5)得到的1M Glucose-Glycine溶液(MRPs(P))；但是兩MRPs溶液經Sephadex G-25 column分離後，卻分出兩種迥然不同的結果；在磷酸鹽緩衝液下加熱得到的MRPs(P)會清楚分成兩帶；其帶一具有最高的褐變指數及280nm吸光值，且具有最強的DNA斷裂活性。而在純水下加熱得到的MRPs(H)則可以分成三帶；帶一具有最高的褐變指數及280nm吸光值，而具有最強DNA斷裂活性的濾液則出現在帶二。且經Blue Dextr
1M glucose-glycine solutions heated at 100℃with various concentrations of phosphate buffer(initial pH 7.5). The results of the reactions indicated that the higher concentration of phosphate buffer used, the higher concentration of MRPs became(Browning index higher). And stronger the DNA Breaking Activity of MRPs produced. They are duplicates of the results of 1M glucose-glycine solutions incubated at 25℃ with various concentrations of phosphate buffer(initial pH 7.5). MRPs(H) produced in 1M glucose-glycine solutions heated at 100℃ in water for 48hr showed higher browning index. But the MRPs(P) produced in 1M glucose-glycine solutions heated at 100℃ with 1M phosphate buffer for few hours still have higher DNA Breaking Activity. There are three buffers(MOPS,BES,and phosphate buffers), when the initial pH value the same, concentrations the same, they have similar buffer capacity; because they have similar pKa value. Use these three buffers heating with 1M glucose-glycine solutions, we found the MRPs which made from 1M glucose-glycine solutions heated with phosphate buffer had the most strong DNA Breaking Activity. Contral the concentration of phosphate buffer, 1M glucose-glycine solutions heated at 100℃with various initial pH values of phosphate buffer(1M). The initial pH value lower, the reaction rate lower(Browning index lower). But DNA Breaking Activity were identical. The MRPs were prepared by refluxing 1M glucose-glycine in 1M phosphate(at 100℃ and pH 7.5) for 1hr and fractionated by membrane filters with various ranges of molecular weight cut off (MWCO). The molecular weight of the most MRPs were below 5,000. This major fraction was chromatograph into two bands by Sephadex G-25 column; and one(band 1) of them showed strong DNA Breaking Activity. By ashing method to analyze total phosphorus of the subfractions from G-25 column was not identical from the band 1 fractions. Most of the total phosphorus were inorganic phosphorus, that indicated the phosphate did not bounded to the MRPs. On the other hand, 1M glucose-glycine solutions heated at 100℃ in water(MRPs(H)) for 24hr、initial pH 7.5, had simillar browning index to that of MRPs heated in 1M phosphate buffer(MRPs(P)) for 1 hour. Two MRPs solutions were fractioned by Sephadex G-25 column, there were produce two differert results. MRPs(P) were separate into two bands; the band 1 had higher browning index, with maximum the absorption of 280nm and the higher DNA Breaking Activity. MRPs(H) were separate into three major bands; the band 1 had best browning index and the absorption of 280nm. But the greatest DNA Breaking Activity was appear in band 2. The fractions of MRPs were compared and investigated by Blue dextran; the molecular weight of the band 1, band 2 of MRPs(P),and band 2,band 3 of MRPs(H) were below 5,000. And the molecular weight of the band 1 of MRPs(H) was above 5,000. This result confirm the MRPs of MRPs(H) and MRPs(P) were different.-1 -aInfluence of the phosphate buffer on DNA breaking activity of maillard reaction products of glucose-glycine system |
