Kozulić B, Leustek I, Pavlović B, Mildner P, Barbarić S
Laboratory of Biochemistry, Faculty of Food Technology and Biotechnology, University of Zagreb, Yugoslavia.
Appl Biochem Biotechnol. 1987 Oct;15(3):265-78. doi: 10.1007/BF02798454.
Each of the three high-mannose type glycoproteins studied, acid phosphatase, invertase, and glucose oxidase, could be specifically cross-linked through its carbohydrate chains. The procedure involves periodate oxidation of carbohydrate residues followed by reaction of the generated aldehyde groups with adipic acid dihydrazide as a cross-linker. The amount and size as well as solubility of the formed polymers could be efficiently controlled by varying the reaction conditions, i.e., the oxidation degree and the concentrations of glycoproteins, cross-linker, and hydrogen ions during the cross-linking reaction. It was found that the quantity and size of polymers increased with oxidation degree and protein concentration and by lowering the pH. When the protein concentration was above and pH below certain values, depending on the glycoenzyme, insoluble polymers formed. The soluble cross-linked polymers retained a high level of original activity, and the minor decrease in specific activity noticed was shown to occur during the periodate oxidation step. The cross-linked glycoenzymes are much more resistant to denaturation by high temperature and by changes in pH, demonstrating the usefulness of this method in preparation of the stabilized glycoprotein derivatives.
所研究的三种高甘露糖型糖蛋白，即酸性磷酸酶、转化酶和葡萄糖氧化酶，均可通过其碳水化合物链进行特异性交联。该方法包括对碳水化合物残基进行高碘酸盐氧化，然后使生成的醛基与己二酸二酰肼作为交联剂反应。通过改变反应条件，即交联反应过程中的氧化程度以及糖蛋白、交联剂和氢离子的浓度，可以有效地控制所形成聚合物的量、大小以及溶解度。结果发现，聚合物的量和大小随着氧化程度和蛋白质浓度的增加以及pH值的降低而增加。当蛋白质浓度高于和pH值低于某些值时（取决于糖酶），会形成不溶性聚合物。可溶性交联聚合物保留了高水平的原始活性，并且观察到的比活性的轻微下降表明发生在高碘酸盐氧化步骤。交联糖酶对高温和pH值变化引起的变性具有更强的抵抗力，证明了该方法在制备稳定的糖蛋白衍生物中的实用性。
