Kitano Hiromi, Miyamoto Takashi, Kawasaki Hideaki
Department of Chemical and Biochemical Engineering, Toyama University, Toyama 930-8555, Japan.
J Colloid Interface Sci. 2004 Nov 15;279(2):425-32. doi: 10.1016/j.jcis.2004.06.076.
Alpha-cyclodextrin (alpha-CD) with an amino group was conjugated to an alpha, omega-dicarboxylated poly(ethylene glycol) (PEG). The inhibition constant (Ki) of the alpha-CD-PEG conjugate for the catalysis by beta-amylase was larger than that of alpha-CD, due to a steric obstruction of the PEG moiety to the binding of alpha-CD moiety to beta-amylase. alpha-CD-PEG was further modified with cystamine (CD-PEG-Cys) or cysteamine methyl disulfide (CD-PEG-MDS), and the disulfide-carrying alpha-CD-PEG was accumulated on a gold surface as a self-assembled monolayer (SAM). The binding of beta-amylase to the alpha-CD-PEG SAM was followed by a decrease in cathodic peak current in the voltammogram of hydroquinone as a probe using a cyclic voltammetry (CV). The beta-amylase bound to the alpha-CD-PEG SAM was desorbed by the addition of free alpha-CD, and the ratio of desorbed beta-amylase from the SAM of alpha-CD-PEG-Cys to the total amount of the enzyme bound to the SAM was 40% whereas that from the alpha-CD-PEG-MDS SAM was 83-85%. The percentage of desorption was increased to 100% by the treatment of the alpha-CD-PEG-MDS SAM-carrying electrode with 2-hydroxyethyldisulfide prior to the immersion in the enzyme solution. Adsorption and desorption processes of beta-amylase to the surface of alpha-CD-PEG-MDS SAM were clearly observed using localized surface plasmon resonance absorption spectroscopy. The binding constant of the enzyme to the surface-confined alpha-CD-PEG was much larger than that to free alpha-CD, probably due to a large local concentration of the alpha-CD moiety on the gold surface.
将带有氨基的α-环糊精（α-CD）与α,ω-二羧基化聚乙二醇（PEG）偶联。由于PEG部分对α-CD部分与β-淀粉酶结合的空间位阻，α-CD-PEG缀合物对β-淀粉酶催化的抑制常数（Ki）大于α-CD。α-CD-PEG进一步用胱胺（CD-PEG-Cys）或半胱胺甲基二硫化物（CD-PEG-MDS）修饰，并且携带二硫键的α-CD-PEG作为自组装单分子层（SAM）堆积在金表面。使用循环伏安法（CV），以对苯二酚作为探针，β-淀粉酶与α-CD-PEG SAM的结合伴随着伏安图中阴极峰值电流的降低。通过加入游离α-CD使结合到α-CD-PEG SAM上的β-淀粉酶解吸，从α-CD-PEG-Cys SAM解吸的β-淀粉酶与结合到该SAM上的酶总量的比例为40%，而从α-CD-PEG-MDS SAM解吸的比例为83 - 85%。在将携带α-CD-PEG-MDS SAM的电极浸入酶溶液之前，用2-羟乙基二硫化物处理后，解吸百分比增加到100%。使用局部表面等离子体共振吸收光谱清楚地观察到β-淀粉酶在α-CD-PEG-MDS SAM表面的吸附和解吸过程。酶与表面受限的α-CD-PEG的结合常数远大于与游离α-CD的结合常数，这可能是由于金表面α-CD部分的局部浓度较高。
