Dear Eraldo,

You should determine the prolidase activity in the serum of the patients and control. Then the level in the patients are compared with those of the control.

Herein the method for the determination of prolidase activity:

Prolidase assay

The serum was diluted 40-fold with 2.5 mmol/l Mn2+, 40 mmol/l Trizma HCl buffer (pH 8.0) and pre-incubated at 37°C for 2 h. The reaction mixture containing 30 mmol/l gly-pro, 40 mmol/l Trizma HCl buffer (pH 8.0) and 100 µl of pre-incubation serum in 1 ml was incubated at 37°C for 30 min. Trichloroacetic acid solution (0.5 ml, 20%) was added to stop the incubation reaction. The proline levels in supernatants were assayed by the method proposed by Myara [15], which is a modification of that of Chinard [16]. The intra-assay coefficient of variability (CV) was 3.8%.

Regarding " There is a standard curve for the activity or just compare the experimental and control.". The answer is that you should do both:

Determination of the activity of prolidase using the colorometric method by Myara or Chinard (see below). In this method you should plot a standard curve and compare the results from the patients and control to this standard.

Clin Chim Acta. 1982 Oct 27;125(2):193-205.

Optimal conditions for prolidase assay by proline colorimetric determination: application to iminodipeptiduria.

Myara I, Charpentier C, Lemonnier A.

Abstract

Prolidase assay was reinvestigated by determining proline, using Chinard's method. Although several authors had previously tested this colorimetric reaction, accurate details regarding enzyme activity were not available. The need for greater sensitivity led to the introduction of several modifications: dialysis was eliminated and the substrate concentration and incubation time were changed. In addition, the reaction mixture was preincubated with Mn2+ for 24 h in order to triple prolidase activity. Color development followed at 90 degrees C, because of partial glycylproline hydrolysis at higher temperatures. The effect of several divalent cations on prolidase activity were tested with and without Mn2+. This modified assay was applied to erythrocytes, plasma and skin fibroblasts from a female patient with iminodipeptiduria.

For better understanding please see Table 1 and Figure 1 in the attached file.

Hoping this will be helpful,

Rafik

Serum prolidase activity and oxidative status in patients with bronchial asthma.

Cakmak A1, Zeyrek D, Atas A, Celik H, Aksoy N, Erel O.

Abstract

Asthma is a disease where there is an accumulation of collagen in the reticular basal membrane of the airway leading to chronic inflammation. The enzyme prolidase plays an important role in the breakdown of collagen and the breakdown of intracellular protein especially in the final stage when peptides and dipeptides contain a high level of proline. To evaluate the relationship between prolidase activity and oxidative status in asthma patients. Comparison was made between 42 patients diagnosed with bronchial asthma and 32 healthy children of similar age and gender. Serum prolidase activity was measured spectrophotometrically. Oxidative status was determined using total antioxidant capacity (TAC) and total oxidant status (TOS) measurement. The prolidase activity of the asthma patient group was statistically significant compared with the control group (P< or =0.001). TAC and TOS levels in the asthma patient group were higher than the control group (P< or =0.001, P< or =0.002, respectively). No correlation was found between the prolidase and oxidative levels of the two groups. A positive correlation was determined between the prolidase activity and TAC in the asthma patient group (P< or =0.001, r=0.501). The prolidase enzyme activity, which plays a role in the collagen turnover, was low in the asthma patients; therefore, their collagen metabolism had undergone a change and this indicates that there may be an effect on the accumulation of collagen in the reticular basal membrane. Moreover, the high level of TOS indicates that these patients were exposed to severe oxidative stress with an increased TAC response.

Regards

http://www.ncbi.nlm.nih.gov/pubmed/19288447

Thanks a lot for the reply !

This substance, Gly-Pro, is bought ready?

Yes. You can purchase it.

Rafik