KAPs Research | Condoleo Portfolio

Keratin-Associated Protein Research

Research Assistant - Hofstra University | June 2022 - December 2022 | Hempstead, NY

Background

Prior to my senior year of high school, I conducted a research project under the guidance of Professor Roche C. deGuzman at Hofstra University. My research centered on keratin-associated proteins (KAPs). For background, during the formation of the hair fiber's cortex, disulfide bonds are formed by a matrix of small globular proteins known as KAPs. The disulfide bonds formed during this process are known to have a significant contribution to the strength of the hair fiber, and studies have demonstrated that the breakage of these bonds have been linked to hair strength weakening by over 50%.

In my project, the objective was to keratin-associated proteins from healthy human fibers and create a solubilized treatment. This treatment would then be applied to previously damaged hair.

Methods

I extracted KAPs through a chemical process from healthy human hair fibers. The hair was subjected to this treatment for 96 hours at 37 °C, and the filtrate was dialyzed against distilled water to remove any present chemicals. Once the filtrate was removed from dialysis, the proteins were no longer soluble. However, by adding thioglycolic acid (TGA), a reduced and solubilized extract named ReKAP was produced.

To characterize the KAPs extract, three different assays were used. Using SDS-Page gel-electrophoresis, the molecular weight of the proteins present in the extract was determined, enabling the classification of KAPs. In the DC protein assay, the KAPs' protein concentration was measured and analyzed through the use standard curve of albumin egg. Lastly, the concentration of melanin was determined via spectrophotometry, where a standard curve using synthetic melanin was employed.

After the production and characterization of ReKAP, it was applied to both untreated and damaged hair. The hair in this project was intentionally damaged by using bleaching and heat-damage treatments. Then, to determine the effect of the treatment on the hair, a mechanical tester was used to assess the hair's tensile strength and strain. Additionally, Scanning Electron Microscopy (SEM) was used to analyze the hair samples.

Dialysis of KAPs Extract

Soluble KAPs Extract with TGA

Microplate Reader for Standard Curve

Application of KAPs

Instron Mechanical Tester

Testing of Hair Samples

Bundling of Hair

Sputter Machine

Samples after Sputter

SEM Machine

Results

Using SDS-PAGE, it was determined that the extraction procedure yielded proteins with low Mw, predominantly 10 kilodaltons, confirming the presence of KAPs and the absence of keratin monomer bands. In addition, the ReKAP solution exhibited intensified KAPs bands, indicating that the precipitates from the extract were in fact KAPs that formed disulfide crosslinks to produce larger insoluble molecules.

SEM-Imaging was also utilized to display the structure of hair fibers prior to and following KAPs Extraction. In the residual hair morphology of KAPs-Extracted hair, significant left-over mass with wrinkled structures and scaly cuticle were still visible when compared to normal hair. This indicated that cortical KAPs and melanin leached from the outer cuticle as a result of the extraction.

Next, a dose-response analysis was performed on bleached hair to determine the optimal concentration of ReKAP. At increasing ReKAP treatment concentrations up to 100 percent, the ultimate tensile strength also increased up to 195.3 MPa from 130.6 MPa, demonstrating a dose-response relationship of ReKAP application, in particular a saturation curve function.

The application of ReKAP significantly increased the ultimate tensile strength of bleached hair from 122.5 MPa to 195.6 MPa. While bleaching and heat damage weakened untreated hair, ReKAP did not statistically strengthen untreated or heat-damaged hair. However, its effect on bleached hair was statistically significant.

The stress-strain curve of multiple replicates is displayed to further illustrate the impact of ReKAP application. In this graph, bleached hair samples, depicted in red, and bleached hair samples with ReKAP application, depicted in blue, exhibit two distinct curve groups, illustrating the increase in strength with ReKAP. However, the ultimate tensile strain did not increase significantly.

Delipidized Hair (Left) and KAPs Extract Residual Hair (Right)

Conclusions

The hypothesis was partially supported as in one group, bleached hair, the KAPs had a positive statistically significant impact on the strength of the hair. However, in the other two groups, untreated hair and heat damaged hair, there was no statistical difference in the strength of the hair. Additionally, there was no statistical significance between the strain in any of the groups. This suggests that the application of KAPs may be used to strengthen damaged hair that may be related to chemical processes.