Sauer G R, Wuthier R E
Department of Chemistry, University of South Carolina, Columbia 29208.
J Biol Chem. 1988 Sep 25;263(27):13718-24.
Fourier transform infrared spectroscopy (FTIR) was used to characterize the organic and mineral phases present during the induction of mineral formation by collagenase-released matrix vesicles (CRMV) during incubation in a synthetic cartilage lymph in vitro. CRMV mineralization, which occurs in the absence of alkaline phosphatase organic phosphate substrates, is characterized by an initial short lag period of limited Ca2+ accumulation, followed by a period of rapid Ca2+ uptake, and finally, by a plateau period during which Ca2+ accumulation continued at a slower rate. FTIR spectra taken at timed intervals during the induction of mineralization revealed the presence of absorptions characteristic of protein, phospholipid, and mineral components in the CRMV. These became progressively more intense with time. To reveal underlying changes occurring during the successive stages of Ca2+ accumulation, FTIR spectra of nascent (or demineralized) CRMV were computer-subtracted from subsequent spectra, nulling on the C-H stretch modes characteristic of the lipid acyl chains. These difference spectra showed little change during early Ca2+ loading, revealing that mineral ions initially accumulated in a form similar to that present in nascent matrix vesicles (MV). During the period of rapid Ca2+ uptake prior to appearance of crystalline mineral, difference spectra revealed subtle changes in the carbonyl and amide nitrogen stretch modes indicative of protein conformational changes. The first definable mineral phase appeared late in the rapid Ca2+ uptake period and was a distinct, crystalline octacalcium phosphate (OCP)-like phase. With time, the OCP-like precursor became more apatitic in character. There was no evidence that any amorphous calcium phosphate phase formed during the MV mineralization sequence. The mature MV mineral phase closely resembled hydroxyapatite formed via an OCP precursor and was similar to other biological apatites that show a substantial incorporation of carbonate.
傅里叶变换红外光谱（FTIR）用于表征在体外合成软骨淋巴液中孵育期间，胶原酶释放的基质小泡（CRMV）诱导矿物质形成过程中存在的有机和矿物质相。CRMV矿化发生在没有碱性磷酸酶有机磷酸盐底物的情况下，其特征是最初有一段短暂的Ca2+积累有限的延迟期，随后是快速Ca2+摄取期，最后是一个平稳期，在此期间Ca2+以较慢的速率继续积累。在矿化诱导过程中按时间间隔采集的FTIR光谱显示，CRMV中存在蛋白质、磷脂和矿物质成分的特征吸收。这些吸收随时间逐渐增强。为了揭示Ca2+积累连续阶段发生的潜在变化，将新生（或脱矿质）CRMV的FTIR光谱从后续光谱中进行计算机减法处理，消除脂质酰链特征性的C-H伸缩模式。这些差异光谱在早期Ca2+加载期间变化很小，表明矿物质离子最初以与新生基质小泡（MV）中存在的形式相似的形式积累。在结晶矿物质出现之前的快速Ca2+摄取期，差异光谱显示羰基和酰胺氮伸缩模式有细微变化，表明蛋白质构象发生了变化。第一个可定义的矿物质相出现在快速Ca2+摄取期后期，是一个独特的、类似结晶八钙磷酸盐（OCP）的相。随着时间的推移，类似OCP的前体在性质上变得更接近磷灰石。没有证据表明在MV矿化序列中形成了任何无定形磷酸钙相。成熟的MV矿物质相与通过OCP前体形成的羟基磷灰石非常相似，并且与其他显示大量碳酸盐掺入的生物磷灰石相似。
