Recombinant Human Adenosine Deaminase Protein: Research Guide

Recombinant Human Adenosine Deaminase Protein is a critical enzyme tool for researchers investigating purine metabolism, immune cell function, and the molecular basis of adenosine deaminase deficiency-related immunodeficiency.

Protein Name

Recombinant Human Adenosine Deaminase (ADA)

UniProt ID

P00813 (ADA_HUMAN) — please verify

Enzymatic Function

Catalyzes the irreversible deamination of adenosine and 2′-deoxyadenosine to inosine and 2′-deoxyinosine, respectively

Biological Relevance

Essential for purine salvage pathway; ADA deficiency causes severe combined immunodeficiency (SCID)

Recommended Storage

-20 °C or -80 °C; avoid repeated freeze-thaw cycles (verify with CoA)

Primary Applications

Enzyme activity assays, immunological research, drug discovery, substrate specificity studies

SKU

rp364763

Protein Overview and Biological Background

Adenosine Deaminase (ADA; EC 3.5.4.4) is a zinc-containing enzyme of the purine salvage pathway responsible for the irreversible hydrolytic deamination of adenosine and 2′-deoxyadenosine to inosine and 2′-deoxyinosine, respectively. In humans, the ADA gene encodes a ~41 kDa monomeric protein that is ubiquitously expressed, with highest activity detected in lymphoid tissues.

Dysregulation or deficiency of ADA leads to the accumulation of deoxyadenosine and its phosphorylated derivatives, which are selectively toxic to lymphocytes, particularly T and B cells. This underlies the pathophysiology of ADA-deficient severe combined immunodeficiency (ADA-SCID), a well-characterized primary immunodeficiency disease. Recombinant Human ADA Protein (SKU: rp364763) provides researchers with a highly pure, biologically active form of this enzyme for in vitro research applications.

Note: Specific molecular weight, purity grade, host expression system, and tag information should be confirmed against the Certificate of Analysis (CoA) supplied with each lot.

Reconstitution, Handling, and Storage

Proper handling of Recombinant Human Adenosine Deaminase Protein is essential to preserve enzymatic activity and ensure reproducible experimental results. Follow the guidelines below:

• Reconstitution: Briefly centrifuge the vial before opening to collect lyophilized material at the bottom. Reconstitute in sterile, endotoxin-free deionized water or the buffer recommended in the product CoA. Allow the solution to equilibrate at room temperature for 5–10 minutes with gentle mixing; avoid vortexing.
• Working Concentration: Prepare aliquots at the working concentration required for your assay. Refer to the CoA for the recommended concentration range.
• Short-term Storage: Store reconstituted protein at 4 °C for up to 1 week when supplemented with a carrier protein (e.g., 0.1% BSA) and a protease inhibitor if necessary.
• Long-term Storage: Aliquot and store at -20 °C or -80 °C. Avoid repeated freeze-thaw cycles, which can significantly reduce enzymatic activity. Addition of glycerol (final concentration 5–10%) may improve stability.
• Sterility and Endotoxin: Confirm endotoxin levels from the CoA before use in cell-based assays to prevent confounding inflammatory responses.

All specific buffer compositions, concentration recommendations, and stability data should be verified against the current lot CoA.

Experimental Applications and Assay Protocols

Recombinant Human Adenosine Deaminase Protein supports a broad range of biochemical and cell biology research applications. Representative uses include:

1. Enzyme Activity Assay

ADA activity is commonly measured by monitoring the conversion of adenosine to inosine via UV spectrophotometry (decrease in absorbance at 265 nm) or by fluorometric detection of ammonia released during deamination. Ensure assay buffer pH is maintained between 7.0 and 7.5 for optimal activity. Include appropriate negative controls (heat-inactivated enzyme) and positive controls (known ADA standard).

2. Substrate Specificity and Kinetic Studies

Use Recombinant Human ADA to determine Michaelis-Menten kinetics (Km and Vmax) for adenosine and 2′-deoxyadenosine. Vary substrate concentrations across a range that spans the expected Km value (verify from literature or internal data). Inhibitor studies with compounds such as erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) or pentostatin may be performed to characterize inhibitor potency (IC50, Ki).

3. Immunological Research

ADA plays a key role in T-cell and B-cell development. Recombinant Human ADA Protein can be used to rescue lymphocyte cultures from deoxyadenosine-mediated toxicity, or as a positive control in studies of ADA-SCID disease modeling. It may also be used in co-culture experiments to assess the paracrine role of extracellular ADA in immune cell signaling.

4. Drug Discovery and Screening

Recombinant Human ADA Protein is suitable as a target enzyme in high-throughput screening (HTS) campaigns for novel ADA inhibitors with potential utility in oncology, autoimmunity, or antiviral research.

Specific protocol conditions (enzyme concentrations, incubation times, temperature) should be optimized empirically for each experimental system.

Quality Control and Regulatory Considerations

Aladdin Scientific's Recombinant Human Adenosine Deaminase Protein (SKU: rp364763) is manufactured and quality-controlled to support research use. Researchers should review the following quality parameters from the product CoA before use:

• Purity: Assessed by SDS-PAGE and/or HPLC; confirm the purity specification meets the requirements of your assay (e.g., ≥90% or ≥95%).
• Biological Activity: Confirmed by a functional enzyme activity assay; specific activity (units/mg) should be stated on the CoA.
• Endotoxin Level: Measured by LAL assay; critical for cell-based and in vivo studies.
• Identity: Confirmed by Western blot or mass spectrometry using anti-ADA antibody or peptide mapping.
• Lot-to-Lot Consistency: Compare CoA data across lots when longitudinal experiments require consistency.

Regulatory Notice: This product is intended for research use only (RUO) and is not approved for diagnostic, therapeutic, or clinical use. Users are responsible for compliance with local regulations governing the use of recombinant proteins in their respective institutions.

Frequently asked questions

What expression system is used to produce Recombinant Human Adenosine Deaminase Protein (rp364763)?

The specific host expression system (e.g., E. coli, insect cell, or mammalian cell) for SKU rp364763 should be confirmed in the product datasheet or Certificate of Analysis (CoA). Different expression systems may affect post-translational modifications and specific activity. Please contact Aladdin Scientific technical support or refer to the product page for the most current lot-specific information.

How should I determine the optimal working concentration of Recombinant Human ADA Protein for my enzyme activity assay?

Optimal working concentration depends on your specific assay format, substrate concentration, detection method, and desired sensitivity. We recommend performing a preliminary titration experiment using a range of enzyme concentrations under your standard assay conditions (pH 7.0–7.5, 37 °C) and selecting the concentration that gives a linear response within the detection range. Refer to the CoA for the stated specific activity as a starting reference point.

Can Recombinant Human Adenosine Deaminase Protein be used in cell-based assays?

Yes, Recombinant Human ADA Protein can be used in cell-based assays, for example to rescue lymphocytes from deoxyadenosine toxicity or to study extracellular ADA signaling. However, it is critical to first verify the endotoxin level from the CoA, as high endotoxin content can confound cell-based results by triggering non-specific inflammatory responses. Use only the minimum effective concentration needed for your experimental endpoint.

What are the recommended conditions for long-term storage of this recombinant protein?

For long-term storage, aliquot the reconstituted protein into single-use volumes to avoid repeated freeze-thaw cycles and store at -80 °C. Addition of a carrier protein (0.1% BSA) and cryoprotectant (5–10% glycerol) is generally recommended to maintain stability. Always verify storage recommendations against the specific lot CoA, as formulation may vary between lots.

Is Recombinant Human Adenosine Deaminase Protein (rp364763) suitable for use as a positive control in ADA-SCID research?

Recombinant Human ADA Protein is widely used in research settings as an enzyme replacement or positive control in studies modeling ADA-deficient SCID. Its use as a positive control is appropriate provided the product demonstrates confirmed biological activity (as stated on the CoA) under your assay conditions. This product is for research use only and is not intended for therapeutic application.

References

1. Giblett ER, Anderson JE, Cohen F, Pollara B, Meuwissen HJ. Adenosine-deaminase deficiency in two patients with severely impaired cellular immunity. Lancet. 1972;2(7786):1067-1069.
2. Hershfield MS. Adenosine deaminase deficiency. In: Adam MP, et al., eds. GeneReviews. Seattle: University of Washington; 1993-2023.
3. Kredich NM, Hershfield MS. S-adenosylhomocysteine toxicity in normal and adenosine kinase-deficient lymphoblasts of human origin. Proc Natl Acad Sci USA. 1979;76(5):2450-2454.
4. UniProt Consortium. UniProt entry P00813 (ADA_HUMAN). Available at: https://www.uniprot.org/uniprot/P00813

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.