Application of KP-Cryst Fusion Proteins

KP-Cryst Wt and KP-Cryst Mut fusion proteins were applied to Asian and Caucasian Brown virgin hair. The hair strands were washed with a classic commercial shampoo (Pantene® Pro-V Classic) before application. For the treatments, 400 mg of each hair type were incubated with the KP-Cryst Wt and KP-Cryst Mut proteins dissolved in a buffer composed by 25 mM HEPES and 120 mM NaCl, at pH5 and pH9. Prior treatment, the hair strands were pre-conditioned with 1 mL of buffer, for 15 min at room temperature, and then dried with the hairdryer. Then, 1 mL of protein solution (1 mg/mL) was applied to the hair strands, incubated for 15 min, and dried with hairdryer. This step was repeated 5 times, with a total 5 mg of protein applied in each hair strand. Hair strands only incubated with the buffer were treated as controls. All samples were thoroughly washed in tap water with the same commercial shampoo and dried with hairdryer 24 h after treatment.

An amount of 20 mg of treated and untreated hair fibers were incubated with 2 mL of 1.04 × 10⁻⁴ M Rhodamine B aqueous solution, pH 4.1, in a bath at 50°C, for 30 min. After incubation, the fibers were rinsed 5 times with deionized water to remove loosely attached dye molecules, and were dried under a nitrogen atmosphere (dos Santos Silva and Joekes, 2005). Individual hair fibers were photographed with a fluorescence microscope (Olympus BX51, Massachusetts USA; excitation = 535/550 nm, emission = 645/675 nm) for the determination of the apparent diffusion coefficient by image analysis using image processing program ImageJ 1.46 r.

For the DSC characterization, 2 mg of each hair samples (with and without the KP-Cryst fusion proteins) were analyzed using DSC instruments (DSC 6000, Perkin Elmer). Thermal studies of KP-Cryst Wt and KP-Cryst Mut proteins were conducted using a power compensated differential scanning calorimetry instrument and aluminum pans (max. pressure: 1 bar), at a temperature range from 50 to 265°C (heating rate: 5°C/min). The DSC instrument calibration was performed using high-purity indium and zinc and all samples were measured in duplicate, with the mean value and standard deviations calculated and presented (Tinoco et al., 2018).

The effect of KP-Cryst Wt and KP-Cryst Mut proteins on the mechanical properties of hair was assessed by the differences in the Young's modulus before and after treatment with the proteins. The hair mechanical properties were determined following guidelines outlined in ASTM D1145-95 for fiber tensile testing. Tensile tests were performed using a Hounsfield dynamometer H100KS Model and a set of 25 hair fibers with low variability was selected for each condition. Each hair was individually mounted in the tensile jig by means of a paper template with a fixed gauge length of 20 mm and placed in an excicator prior to the analysis. A load range of 25 N and a speed of 1.5 mm/min were defined as settings for the tensile strength test. For each hair, applied load against extension were recorded and, using an average mean diameter of 70 μm, the data were converted to stress (load/unit area) vs. strain (% extension). All measurements were made in the middle part of the hair fiber (Tinoco et al., 2018).