Measuring Binding Constants of His-Tagged Proteins Using Affinity Chromatography and Ni-NTA Immobilized Enzymes

2020 ◽  
pp. 405-416
Author(s):  
Annette C. Moser ◽  
Benjamin White ◽  
Frank A. Kovacs
Keyword(s):  
Affinity Chromatography ◽  
Binding Constants ◽  
Immobilized Enzymes

Antibody to spermine: a natural biological constituent

Science ◽  
1980 ◽  
Vol 208 (4448) ◽  
pp. 1178-1181 ◽  
Author(s):  
D Bartos ◽  
F Bartos ◽  
RA Campbell ◽  
DP Grettie ◽  
P Smejtek
Keyword(s):  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Binding Constants ◽  
Cross Reactivity ◽  
Polyamine Metabolism ◽  
Regulatory Function ◽  
Rabbit Serum ◽  
Normal Rabbit Serum ◽  
Immunodiffusion Test ◽  
Gradient Gel Electrophoresis

A protein that binds spermine specifically was separated from normal rabbit serum by affinity chromatography. Immunoelectrophoresis, the Ouchterlony immunodiffusion test, and gradient gel electrophoresis indicated that this protein has immunoglobulin characteristics and consists of several populations of antibodies to spermine. These were sequentially released from Sepharose-spermine gel by step-wise elution with solutions ranging in pH from 4 to 1. The binding constants varied from 5.0 x 10(8) to 11.1 x 10(8) liters per mole. These globulins did not react with monoacetylputrescine, L-ornithine, L-lysine, and histamine. Negligible cross-reactivity was detected with spermidine, putrescine, N8-monoacetylspermidine, cadaverine, and diaminopropane. Since perturbations in polyamine metabolism have been identified in several diseases, the study of extracellular polyamine homeostasis may reveal an important regulatory function for this protein.

scholarly journals Calcium-binding constants of trypsin and trypsinogen. A reassessment

1981 ◽  
Vol 193 (2) ◽  
pp. 655-658 ◽  
Author(s):  
S G R Cliffe ◽  
D A W Grant
Keyword(s):  
Affinity Chromatography ◽  
Calcium Binding ◽  
Cation Exchanger ◽  
Binding Constants ◽  
High Affinity ◽  
High Affinity Site ◽  
Logarithmic Unit

The Ca2+-binding constants for trypsin and trypsinogen have been reassessed by using enzyme that has been purified by affinity chromatography and measuring the distribution of 45Ca2+ between the protein and a cation exchanger. The pKCa2+ value of 4.5 for the high-affinity site on trypsin was 1 logarithmic unit greater than that previously reported.

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