AFM visualization of immobilized horseradish peroxidase during its catalytic cycle

Using atomic force microscopy (AFM), the heights of individual horseradish peroxidase (HRP) molecules were determined at different stages of the catalytic cycle of this enzyme (I-IV). The HRP heights were determined in two buffer systems: Dulbecco's phosphate-buffered saline (PBSD) (pH 7.2) and citrate-phosphate buffer (CPB) (pH 5.0). Spectrophotometric analysis showed that the enzyme was more active in CPB. AFM data processing under these conditions revealed a tendency toward a decrease in the HRP molecule height after addition of the ABTS substrate (stage (II)). After the addition of H2O2 (stage (III)), an increase in the height was observed followed by a further decrease after the addition of the NaN3 inhibitor (stage (IV)). No such trend was observed in PBSD. The increase in the molecular height at stage (III) (in the presence of all enzyme system components) was interpreted as an evidence of the HRP activity. The proportion of molecules with the increased height at stage (III) reached 50.5% in CPB and did not exceed 14.8% in PBSD. These data support the existence of "active" and "non-active" HRP molecules in the studied sample, thus confirming the need to move from traditional biochemical "enzyme activity" to identifying "active" and "non-active" enzyme molecules using a fundamentally new approach of analysis of the properties of individual molecules.