If ovalbumin solutions are exposed to elevated temperatures, then the sample containing glycerol will produce the lowest turbidity using the spectrophotometer compared to sucrose, glucose, histidine, and the control, because glycerol most effectively stabilizes protein structure by the mechanism of preferential exclusion and increasing the solution’s viscosity to form a hydrated and protective layer around the protein.

Albumin Based Drugs

Albumin, a type of globular protein, is the most abundant protein in blood plasma. In our bodies, it is more specifically known as human serum albumin (HSA); it is synthesized and secreted into our bloodstream by hepatocytes, or liver cells, to maintain oncotic pressure and prevent fluids from leaking out of our blood vessels. This protein is also responsible for the transportation of bound ligands, which include a variety of substances classified as endogenous ligands, such as fatty acids, bilirubin, calcium, and thyroxine; as well as exogenous ligands such as drugs and toxins.

Due to its binding and transport capacity, albumin has adopted new clinical applications in medical drug delivery. Albumin-based drugs use medical-grade human albumin as a drug delivery carrier, fusion partner, or nanoparticle to enhance drug distribution throughout the body. Certain albumin-binding drug additives, such as fatty acids, mimic endogenous albumin-binding ligands, which allows for greater albumin affinity and increases the likelihood of drug effectiveness. Albumin also has applications as a biocomponent for drugs that help with conditions such as hypoalbuminemia, sepsis, and acute respiratory distress syndrome (ARDS). Albumin-based drug delivery systems also show a promising future for carrying therapeutics for diabetes, cancer, and other diseases.  

Albumin is relatively more heat resistant than other plasma proteins, making albumin-based drugs more capable of maintaining stability. However, they also have their own thermal limits. Most pharmaceutical-grade albumin is stored in temperatures ranging from 2℃ to 25℃ to ensure long shelf life. These drugs are technically resilient to room temperatures and being left outside a proper storing unit, but the repeated thermal fluctuations of the outside environment could cause improper refolding of the protein after being in a higher temperature. Even without temperature fluctuations, when albumin is at temperatures above 30℃ for a long enough period of time, the protein starts degrading because of the rapid movement of particles, causing it to unfold. Other than that, if the seal is damaged on a substance containing albumin, room temperature promotes bacterial growth within the drug, causing it to no longer be safe for use.

In some drugs, where albumin is used as an albumin nanoparticle, it acts as a drug carrier and delivers the drug throughout the body. Their delivery function is quite unique; they have targeted delivery systems, allowing the drug to attack the harmful cells rather than the healthy ones. However, even though this specific usage of albumin is extremely useful, it is actually more sensitive to higher temperatures than regular human serum albumin. After a few hours in an environment over 30℃, it aggregates quickly, much faster than HSA. This reflects the importance of proper storage of these substances, and also how easily we can accidentally cause thermal denaturation to albumin by leaving it out in the open, through thermal radiation, protracted transportation without proper insulation, and many more. For the sake of shelf life and medical safety, advancements need to be made for the prevention of aggregation and the increase of stability.

Thermal Denaturation

Denaturation of proteins refers to the unfolding of proteins that happens as a result of the damaging of the secondary and tertiary bonds of the proteins from stressors. Proteins maintain their globular shape through interactions between hydrogen bonds, hydrophobic and ionic interactions, and van der Waals forces. During thermal stress induced denaturation, kinetic energy  and molecular motion disrupts the hydrogen bonds of a protein and causes a loss of the protein globular structure. This turns the three-dimensional shape of the protein into a less ordered form, often leading to a loss of proper function, as its job depends on its structural shape. 

As proteins unfold, hydrophobic regions normally inside the protein are revealed, these regions tend to interact with other hydrophobic regions and cause the proteins to clump together to form stable, insoluble structures. This leads to aggregation, in which misfolded proteins form amorphous clumps. Aggregation is generally irreversible once the clumps form and are usually the subsequent event of denaturation. Generally, the higher the temperature, the greater the rate of denaturation and aggregation. 

Protein denaturation and aggregation can be measured through turbidity, or the cloudiness of a liquid. When aggregates form, a spectrophotometer can measure how much light is scattered by the suspended particles within the liquid, detecting the optical density of the solution at wavelengths typically within the 400-600nm range. A higher turbidity reading indicates greater aggregation and greater cloudiness.

Preventing protein denaturation in drugs is significant because once the protein loses the original shape, it becomes a critical quality and safety issue in the drug. Structural changes also impact drug efficacy and function of the drugs. Protein aggregates may also trigger unwanted immune responses as it may induce the formation of anti-drug antibodies that neutralize the therapeutic effects of the drug. Therefore, understanding and minimizing changes to the proteins in biotherapeutics prevents adverse patient reactions and fatalities.

Protein Stabilizers 

Thermal protein stabilizers are substances that are added to proteins that help prevent denaturation and aggregation under thermal stress. These stabilizers work in a variety of ways and can be categorized into three main types: sugars, sugar alcohols, and amino acids.

Sugars act as thermal protein stabilizers through the mechanism of preferential exclusion. This is a process in which co-solvents, such as sugar, are excluded from the protein's immediate hydration shell instead of binding to the protein. Sugars are more strongly attracted to water than the protein’s surface, creating  a layer of water surrounding the protein. The system favours the most compact protein structure with the smallest surface area, stabilizing the protein by maintaining its natural conformation and minimizing conformational mobility even under thermal stress. 

Sugar alcohols work to stabilize proteins through a combination of preferential exclusion (similar to sugars) and water structure modification. They act as “water structure makers” by reducing the number of free water molecules and increasing the viscosity of the solution. This forms a hydrated, protected layer around the proteins which helps prevent denaturation from thermal stress.

Amino acids also use preferential exclusion as a way to prevent protein denaturation. Additionally, some amino acids are able to bind to the hydrophobic areas of a partially unfolded protein. These hydrophobic patches, usually situated in the interior of the natural protein, become exposed as it unfolds. By binding to these exposed and charged areas, the amino acid shields them from the aqueous solution and prevents the intermolecular interactions that cause them to stick together and aggregate. Thus, reducing the rate at which proteins unfold when exposed to higher temperatures.

Overall, thermal protein stabilizers alter the environment in which the proteins are located to make the unfolding of proteins thermodynamically unfavourable.

Ovalbumin

Ovalbumin is the primary protein found in egg whites, accounting for 54% of the protein content. It is also the model protein we selected for this experiment. Similar to albumin, it is a globular, water soluble protein known to undergo thermal denaturation and aggregation, coagulating at approximately 80℃. Although ovalbumin is not entirely identical to albumin, they share key characteristics and reactions to thermal stress. They are also both commonly used in experiments to model protein denaturation. Due to its accessibility, safe, and clear aggregation behavior, ovalbumin serves as an appropriate experimental model for this investigation. However, it is essential to note that the molecular differences between ovalbumin and albumin proteins means that results can only provide insights into general stabilization trends of albumin rather than exact reflections of how albumin would behave.

Manipulated variable: stabilizer used, temperature

Controlled variable: time, pH, ovalbumin, usage of water bath

Responding variable: turbidity of solution

Materials: 

• Test tubes
• Scale 
• Graduated cylinder
• Water Bath
• Spectrophotometer 
• Pipettes
• Cuvette 
• Stabilizers:
  • Sucrose (sugar) 10%
  • Glucose (sugar) 10%
  • Glycerol (sugar alcohol) 10%
  • Histidine (amino acid) 50 mM (0.8%) 
• Egg Whites (1:20 ratio)
• Neutral pH buffer solution

Preparation 

1. Wear PPE

Solution Preparation Buffer solution

1. Prepare 20 mM of phosphate buffer 

Sucrose stock solution (20% w/v)

1. Weigh 10 g of sucrose in a beaker
2. Add buffer solution until the total volume reaches 50 mL
3. Mix until fully dissolved

Glucose stock solution (20% w/v)

1. Weigh 10 g of glucose in a beaker
2. Add buffer solution until the total volume reaches 50 mL
3. Mix until fully dissolved

Glycerol stock solution (20% v/v)

1. Measure 10 mL in a beaker
2. Add buffer solution until the total volume reaches 50 mL
3. Mix until fully dissolved

Histidine stock solution (100 mM)

1. Weigh 0.775  g of histidine in a beaker
2. Add buffer solution until the total volume reaches 50 mL
3. Mix until fully dissolved

Egg White

1. Separate egg whites from yolk
2. Filter through to remove membranes or clumps
3. Measure 10 mL of egg white and 190 mL of buffer solution to form a 1:20 dilution
4. Gently mix until fully dissolved

Lab Procedure

1. Add 5 mL of ovalbumin solution to each of the test tubes
2. To each test tube, add 5 mL of each type of stabilizer solution, adding pH buffer solution to one of the test tubes as a control
3. Place each test tube into the water bath for 30 minutes
4. In 10 minute intervals, take the test tubes out and measure/record its turbidity using the spectrophotometer

Due to time constraints and lab scheduling difficulties, we were unable to conduct the experiment prior to the online platform due date March 3rd, 2026. For the observations from our experiment, please visit this link: https://docs.google.com/document/d/1fCntz22WcNXqO2c1jIiqLnJ5Zdo4vlDDvvNIaqrATyE/edit?usp=sharing

For the analysis from our experiment, please visit this link: https://docs.google.com/document/d/1fCntz22WcNXqO2c1jIiqLnJ5Zdo4vlDDvvNIaqrATyE/edit?usp=sharing

For the conclusions from our experiment, please visit this link: https://docs.google.com/document/d/1fCntz22WcNXqO2c1jIiqLnJ5Zdo4vlDDvvNIaqrATyE/edit?usp=sharing

For the applications for this project, please visit this link: https://docs.google.com/document/d/1fCntz22WcNXqO2c1jIiqLnJ5Zdo4vlDDvvNIaqrATyE/edit?usp=sharing

For the sources of error from our experiment, please visit this link: https://docs.google.com/document/d/1fCntz22WcNXqO2c1jIiqLnJ5Zdo4vlDDvvNIaqrATyE/edit?usp=sharing

Ajito, S., Iwase, H., Takata, S., & Hirai, M. (2018). Sugar-Mediated Stabilization of Protein against Chemical or Thermal Denaturation. The Journal of Physical Chemistry B, 122(37), 8685–8697. https://doi.org/10.1021/acs.jpcb.8b06572

Albumin Guide: Hydration\, Nutrition\, and liver Function | Learn with Superpower. (n.d.). https://superpower.com/biomarker-guides/albumin?srsltid=AfmBOop3fq85UV-BmFC-3x5-9yJ1Y3p-AOE7RF5SRbYX2gv29AK7CwxG

Daniels, A., PhD. (2026, January 9). Why Analyze Protein Aggregates in Formulation Development? YOKOGAWA. https://www.fluidimaging.com/blog/why-is-protein-aggregate-analysis-important-during-formulation-development

Explainer: What is Albumin? (2024, March 25). CSL. https://www.csl.com/we-are-csl/vita-original-stories/2020/explainer-what-is-albumin Hornok, V. (2021). Serum albumin nanoparticles: Problems and prospects. Polymers, 13(21), 3759. https://doi.org/10.3390/polym13213759

Karimi, M., Bahrami, S., Ravari, S. B., Zangabad, P. S., Mirshekari, H., Bozorgomid, M., Shahreza, S., Sori, M., & Hamblin, M. R. (2016). Albumin nanostructures as advanced drug delivery systems. Expert Opinion on Drug Delivery, 13(11), 1609–1623. https://doi.org/10.1080/17425247.2016.1193149

Kendrick, B. S., Chang, B. S., Arakawa, T., Peterson, B., Randolph, T. W., Manning, M. C., & Carpenter, J. F. (1997). Preferential exclusion of sucrose from recombinant interleukin-1 receptor antagonist: Role in restricted conformational mobility and compaction of native state. Proceedings of the National Academy of Sciences, 94(22), 11917–11922. https://doi.org/10.1073/pnas.94.22.11917

Kuroiwa, I., Maki, Y., Matsuo, K., & Annaka, M. (2024). Protein preferential solvation in (Sucralose + water) mixtures. The Journal of Physical Chemistry B, 128(3), 676–683. https://doi.org/10.1021/acs.jpcb.3c06317

Moman, R. N., Gupta, N., & Varacallo, M. A. (2022, December 26). Physiology, Albumin. StatPearls - NCBI Bookshelf. https://www.ncbi.nlm.nih.gov/books/NBK459198/

Politi, R., Sapir, L., & Harries, D. (2009). The impact of polyols on water structure in solution: a computational study. The Journal of Physical Chemistry A, 113(26), 7548–7555. https://doi.org/10.1021/jp9010026

Qu, N., Song, K., Ji, Y., Liu, M., Chen, L., Lee, R., & Teng, L. (2024). Albumin Nanoparticle-Based Drug Delivery Systems. International Journal of Nanomedicine, Volume 19, 6945–6980. https://doi.org/10.2147/ijn.s467876

Sun, W. Q., & Luo, Y. (2025). Efficacy of sugar alcohols and sugars in protein stabilization during freezing, freeze-drying, and air-drying. Frigid Zone Medicine, 5(2), 65–72. https://doi.org/10.1515/fzm-2025-0007

Header Image: https://www.shutterstock.com/image-illustration/albumin-drug-therapeutics-digital-3d-600nw-2458946521.jpg

We would like to thank Ms. Heron for organizing the science fair at our school, Ms. Foisy for taking time to supervise our experiment and provide guidance, and Ms. Maryam for providing materials and teaching us how to use the spectrophotometer! We also used forms of AI just as ChatGPT to help with brainstorming and refining certain areas of this project.