Please help. An explanation will be very helpful. Thank you **Title: Understanding the Biochemical Processing of Oxytocin****Introduction:**Oxytocin is a 9-residue peptide hormone involved in signaling pathways related to reproduction and perinatal health. Initially produced as a proprotein, oxytocin is synthesized as Oxytocin-neurophysin I (proOT-NPI), which is further processed into functional proteins.**Diagram Overview:**1. **Initial Processing:** - ProOT-NPI undergoes processing via **Enzyme 1** to split into proOT and neurophysin I (NPI). - The initial structure includes a polypeptide chain extended by a polyhistidine tag (6xHis) at the C-terminus.2. **Further Processing:** - ProOT undergoes further processing using **Enzymes 2 and 3** to yield active oxytocin (OT). - This step involves the removal of a Lys-Arg dipeptide.**Purification Task:**You are tasked with purifying mature OT from a cell lysate expressing proOT-NPI with a C-terminal polyhistidine tag. The molecular weight (MW) and pI values for proOT-NPI, NPI, proOT, and OT are as follows:| Protein | MW (Da) | pI ||-------------|---------|------|| ProOT-NPI | 10593 | 4.94 || NPI | 9600 | 4.94 || ProOT | 1010 | 5.51 || OT | 1007 | N/A* |*Oxytocin is modified to remain neutral except for the ionizable phenolic OH of tyrosine (pKᵃ=10).**Purification Strategy:**- **Assumptions:** - Conversion reactions may be incomplete; purification from starting material may be required. - Compounds must differ by at least 1000 Da for separation using size-exclusion chromatography. - A pI difference of 1 is necessary for ion exchange chromatography.- **Approach:** - Employ biochemical equipment and purified processing enzymes discussed in class. - Design a systematic purification scheme, possibly incorporating a drawing, and explain each step's rationale.Understanding and implementing these strategies will aid in the efficient purification of oxytocin, ensuring its functionality for physiological applications.

Answered: proOT NP I J Processing V Eneymes Ž:3…

Biochemistry

9th Edition

ISBN:9781319114671

Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Chapter1: Biochemistry: An Evolving Science

Section: Chapter Questions

Problem 1P

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Please help. An explanation will be very helpful. Thank you

**Title: Understanding the Biochemical Processing of Oxytocin**

**Introduction:**
Oxytocin is a 9-residue peptide hormone involved in signaling pathways related to reproduction and perinatal health. Initially produced as a proprotein, oxytocin is synthesized as Oxytocin-neurophysin I (proOT-NPI), which is further processed into functional proteins.

**Diagram Overview:**
1. **Initial Processing:**
- ProOT-NPI undergoes processing via **Enzyme 1** to split into proOT and neurophysin I (NPI).
- The initial structure includes a polypeptide chain extended by a polyhistidine tag (6xHis) at the C-terminus.

2. **Further Processing:**
- ProOT undergoes further processing using **Enzymes 2 and 3** to yield active oxytocin (OT).
- This step involves the removal of a Lys-Arg dipeptide.

**Purification Task:**
You are tasked with purifying mature OT from a cell lysate expressing proOT-NPI with a C-terminal polyhistidine tag. The molecular weight (MW) and pI values for proOT-NPI, NPI, proOT, and OT are as follows:

| Protein | MW (Da) | pI |
|-------------|---------|------|
| ProOT-NPI | 10593 | 4.94 |
| NPI | 9600 | 4.94 |
| ProOT | 1010 | 5.51 |
| OT | 1007 | N/A* |

*Oxytocin is modified to remain neutral except for the ionizable phenolic OH of tyrosine (pKᵃ=10).

**Purification Strategy:**
- **Assumptions:**
- Conversion reactions may be incomplete; purification from starting material may be required.
- Compounds must differ by at least 1000 Da for separation using size-exclusion chromatography.
- A pI difference of 1 is necessary for ion exchange chromatography.

- **Approach:**
- Employ biochemical equipment and purified processing enzymes discussed in class.
- Design a systematic purification scheme, possibly incorporating a drawing, and explain each step's rationale.

Understanding and implementing these strategies will aid in the efficient purification of oxytocin, ensuring its functionality for physiological applications.

Expert Solution

Step 1

PrO-OT-NPI has six Histidine residues in the tag form. Processing enzyme I convert it into Pro-Ot and NPI. There is a large difference between the molecular weights of Pro-OT and NPI. Now by processing enzymes 2 and 3, Pro-OT will be converted to OT or active form of oxytocin. OT is present in the neutral form except at one residue which is phenolic group of tyrosine, it means its PI value is around neutral pH. So, Pro-OT and OT has a difference of more than 1 between their PI values.

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