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DEAE-cellulose chromatography of creatine kinase isoenzymes--effect of pH and serum
Insights
Researchers used DEAE-cellulose chromatography to separate creatine kinase (CK) isoenzymes. Heart (CK2) and brain (CK1) isoenzymes showed distinct elution patterns, with serum albumin affecting CK2 affinity at pH 8.0.
Area of Science:
- Biochemistry
- Protein Chemistry
Background:
- Creatine kinase (CK) exists as multiple isoenzymes, primarily CK1 (brain), CK2 (heart), and CK3 (skeletal muscle).
- Understanding the separation and behavior of these isoenzymes is crucial for diagnosing various medical conditions.
Purpose of the Study:
- To characterize the elution profiles of human heart creatine kinase isoenzymes using DEAE-cellulose chromatography.
- To investigate the influence of pH and serum components on the chromatographic behavior of CK isoenzymes.
Main Methods:
- DEAE-cellulose ion-exchange chromatography was employed to separate human heart creatine kinase isoenzymes.
- Experiments were conducted at different pH values (7.0 and 8.0) and in the presence of serum proteins and albumin.
- Elution conductivity was measured to determine isoenzyme separation.
Main Results:
- Three CK isoenzymes were detected in human heart extracts; CK3 was not retained at pH 7.0.
- CK2 (heart) and CK1 (brain) isoenzymes eluted at specific conductivity values, which varied slightly with pH.
- Serum albumin significantly decreased the affinity of CK2 for DEAE-cellulose at pH 8.0, causing earlier elution, independent of protein aggregation.
Conclusions:
- DEAE-cellulose chromatography effectively separates human creatine kinase isoenzymes based on their charge properties.
- The interaction of CK2 with DEAE-cellulose is sensitive to pH and the presence of serum albumin, suggesting potential implications for in vivo behavior or diagnostic assays.
Abstract:
DEAE-cellulose chromatography (pH 7.0) of human heart extracts revealed the presence of three creatine kinase isoenzymes. The CK3 (skeletal muscle) isoenzyme was not retained on the column under these conditions. The CK2 (heart) and CK1 (brain) isoenzymes eluted at a conductivity of 5.5 +/- 0.6 m omega-1 and 11.4 +/- 1.2m omega-1, respectively. When DEAE-cellulose chromatography was performed at pH 8.0, CK2 eluted at a slightly higher conductivity, 6.5 m omega-1, whereas CK1 eluted as before 12.0 m omega-1. DEAE-cellulose chromatography of CK2 and CK1 isoenzymes in the presence of serum protein, and serum albumin had no significant effect on the elution of CK2 at pH 7.0 and 7.4, and on the elution of CK1 at pH 7.0 and 8.0 However, serum and serum albumin decreased the affinity of CK2 for DEAE-celluose at pH 8.0, and caused this isoenzyme to elute at a conductivity of 3.0-3.5 m omega-1. The decreased affinity of CK2 for DEAE-cellulose was not due to aggregation of CK2 with albumin or some other serum protein, but was related to the amount of albumin applied to the column.
