You can wash your matrix with the buffer and acquire an absorption spectrum, The absobance at 280 nm will give you a measure of the unbound protein.

@Luca ronda..if we wants to leakage for a period of one month..then what will do?Either incubate whole preparation in desired buffer and collect supernatant after 1 day and so on  and read at 280 nm on daily basis and compare with zero days.i think this will give leakage efficiency for a perticular immobilization.

I think that it will be a good way to have a leakage kinetic, if your matrix can stay in a buffer for a month.

thanks luca

Be careful with a quantification at 280nm, it's not necessarilly sensible for low concentrations of enzymes, mostly if you're working with little concentration of enzyme immobilized (µg-mg). Personnally, i prefere to realize a range of enzymatic activity (Vi) (for one substrate  known) depending of the quantity of enzyme in solution (with different concentration of enzyme).

You take the solution with enzyme leaked and you measure the enzyme activity in the same conditions, and you compare with your range to determine the enzyme concentration. It can be a answer more precise than a measure at 280nm. good luck 

the evaluation of enzyme activity can allow to quantify protein samples less concentrate than 280 reading, but you have to assume that the protein activity remains the same after immobilization and leaking, and it needs to be demonstrated. 

Maybe the two tests can be coupled.

I agree with Kalim. To measure the leakage, The enzyme can be soaked in known volume of buffer and supernatent buffer samples can be periodically withdrawn to measure the activity and determine protein concentration by standard method. This values can be quantified for entire volume of buffer added and which would clearly give  a picture of enzyme leaking out of support. Good luck

you may i) use one of the several commercially available colorimetric assays methods (BCA, Bradford, Lowry...), ii)measure absorbance at 280nm, iii)measure enzyme activity of the obtained solution, iv) if the sample is too diluted you may try to concentrate it through freeze drying and then proceed with one of the proposed strategies.  

You can measure repetitive continuos activity cycles, also protein content in supernatant or soaking liquid.

I agree with Filipe Carvalho. The method that should used is depending on the enzyme, the support, the immobilization method , and the pH of the buffer used for enzyme storage; some times we can determine the activity and not protein due to the lower protein concentration.