How long must a species last for, for it be detectable through typical absorbance spectroscopy.
Say I have an enzyme with a turnover number of 300. Each reaction would take only 3.3 milli seconds. An intermediate would exist for only a fraction of that maximal possible time of 3.3 ms.
would it be possible to detect the intermediate during the enzymes reaction using standard absorbance spectroscopy? Would it be possible if the change of the intermediate to the product is not the rate limiting step (in fact its regarded as kinetically insignificant)? I have argued that it would not be.
I know fluorescence has lifetimes in the nanosecond range but here the fluorophore is excited and then emits very quickly and repeatedly and since the light beam is the cause of its transient existence it will happen in sync. In the case of absorbance the intermediate would have to be excited during its transient existence?
A transient intermediate may be detectable by pre-steady-state kinetics methods, requiring a stopped-flow instrument for milli-second reaction times, if its concentration is high enough to have a measurable absorbance at a certain time after the start of the reaction, and if there is a distinct absorbance signature associated with it.
As rightly said, using stopped flow apparatus one can measure the absorption spectra of an intermediate within few milliseconds. This technique is especially useful to study intermediates in fast reactions where the mixing time is comparable to the reaction completion time. If the intermediate is fluorescent then even fluorescence spectra can be measured.
A stopped flow was not used in the experiment. That is why I dislike the results.
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