{"title":"Effect of Chemical Modification on the Distribution of Electrophoretic Mobilities of Individual Molecules of E. coli β-Galactosidase","authors":"Brynne K Riehl, Allyster Klassen, Sumaiya Abas, Winner Pathak, Doug Craig","doi":"10.1139/cjc-2024-0039","DOIUrl":null,"url":null,"abstract":"Escherichia coli β-galactosidase was labelled with 1 mM fluorescein 5-carbamoylmethylthiopropanoic N-hydroxysuccinimidyl ester for 1 and 3 min. The samples were separated by capillary electrophoresis and peak areas compared to that of labelled BSA for the purpose of quantification of the concentration of attached label. Enzyme concentration in the samples was determined by single molecule counting. The average number of labels attached to each molecule of enzyme was found to be 3.1 and 4.5 when labelled for 1 and 3 min respectively. The distribution of single enzyme molecule electrophoretic mobilities for the unlabelled enzyme and that labelled for 1 and 3 min were measured using capillary electrophoresis. The average mobilities were found determined to be -(1.99 ± 0.13) x10-8 m2V-1s-1 (N=39), -(2.16 ± 0.19) x10-8 m2V-1s-1 (N=46), and –(2.18 ± 0.21) x10-8 m2V-1s-1 (N=39) respectively. A protein electrophoresis model was applied and predicted that the differences in average mobilities could be explained through relatively minor changes in overall charge, Stokes radius, and shape. This difference was similar to the range in mobilities observed in the unlabelled protein. This is consistent with the electrophoretic heterogeneity of the unmodified enzyme being caused by relatively small differences in charge, size, and shape of the individual molecules in the population.","PeriodicalId":9420,"journal":{"name":"Canadian Journal of Chemistry","volume":null,"pages":null},"PeriodicalIF":1.1000,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Canadian Journal of Chemistry","FirstCategoryId":"92","ListUrlMain":"https://doi.org/10.1139/cjc-2024-0039","RegionNum":4,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"CHEMISTRY, MULTIDISCIPLINARY","Score":null,"Total":0}
引用次数: 0
Abstract
Escherichia coli β-galactosidase was labelled with 1 mM fluorescein 5-carbamoylmethylthiopropanoic N-hydroxysuccinimidyl ester for 1 and 3 min. The samples were separated by capillary electrophoresis and peak areas compared to that of labelled BSA for the purpose of quantification of the concentration of attached label. Enzyme concentration in the samples was determined by single molecule counting. The average number of labels attached to each molecule of enzyme was found to be 3.1 and 4.5 when labelled for 1 and 3 min respectively. The distribution of single enzyme molecule electrophoretic mobilities for the unlabelled enzyme and that labelled for 1 and 3 min were measured using capillary electrophoresis. The average mobilities were found determined to be -(1.99 ± 0.13) x10-8 m2V-1s-1 (N=39), -(2.16 ± 0.19) x10-8 m2V-1s-1 (N=46), and –(2.18 ± 0.21) x10-8 m2V-1s-1 (N=39) respectively. A protein electrophoresis model was applied and predicted that the differences in average mobilities could be explained through relatively minor changes in overall charge, Stokes radius, and shape. This difference was similar to the range in mobilities observed in the unlabelled protein. This is consistent with the electrophoretic heterogeneity of the unmodified enzyme being caused by relatively small differences in charge, size, and shape of the individual molecules in the population.
期刊介绍:
Published since 1929, the Canadian Journal of Chemistry reports current research findings in all branches of chemistry. It includes the traditional areas of analytical, inorganic, organic, and physical-theoretical chemistry and newer interdisciplinary areas such as materials science, spectroscopy, chemical physics, and biological, medicinal and environmental chemistry. Articles describing original research are welcomed.